Ultra-small prussian blue nano-particles carrying tirofian, preparation method and application of nano-particles in preparation of medicine for reducing MVO and MIRI

Abstract

The invention relates to ultra-small prussian blue nano-particles (T-USPB-C) carrying tirofian, a preparation method of the nano-particles and application of the nano-particles in preparation of drugs for reducing MVO and MIRI. According to the preparation method, the T-USPB-C is prepared by three steps. The T-USPB-C provided by the invention can carry tirofiban, and has catalase and superoxide dismutase simulated nano-enzyme activity. Moreover, the size of the nano-scale drug is required to reach a nano-scale size, and the drug can be efficiently cleared through kidney excretion, so that the potential long-term toxicity problem is minimized. The ultra-small prussian blue nanoparticle carrying the tirofian has an excellent active oxygen scavenging capability. Microvascular perfusion can be rapidly improved through the antithrombotic effect, then the protection effect is continuously achieved through the anti-oxidation and anti-inflammatory mechanism, and microvascular injury and MIRI are effectively prevented.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cardiovascular disease drug preparation, in particular to a kind of carrying tirofiban super small prussian blue nanoparticles, preparation method and its purposes in the preparation of the drug for reducing MVO and MIRI. BACKGROUND

[0002] In patients with myocardial infarction undergoing percutaneous coronary intervention (PCI), epicardial flow recovery is often hindered by coronary microvascular obstruction (MVO), which manifests as slow flow or no-reflow in 10-29% of cases. This triggers myocardial ischemia-reperfusion injury (MIRI) through a vicious cycle of microthrombosis and oxidative stress. Current treatment strategies, including the potent GPIIb / IIIa antagonist tirofiban, only partially alleviate symptoms but carry the risk of systemic bleeding. These processes are interconnected through the "oxidative stress-microthrombosis-inflammation" triad. This vicious cycle is the underlying pathophysiological mechanism of MVO and MIRI, requiring therapeutic strategies that can simultaneously address microvascular obstruction, oxidative stress, and inflammation. The main drug currently used to prevent microvascular obstruction (MVO) is tirofiban, a potent glycoprotein IIb / IIIa (GP IIb / IIIa) receptor antagonist that effectively inhibits the final common pathway of platelet activation, adhesion, and aggregation. Although tirofiban can reduce distal embolization and improve microvascular perfusion, its clinical application is limited due to bleeding complications associated with systemic administration.

[0003] Therefore, in view of the deficiencies of the prior art, it is necessary to provide a kind of carrying tirofiban super small prussian blue nanoparticles, preparation method and its purposes in the preparation of the drug for reducing MVO and MIRI to solve the deficiencies of the prior art. SUMMARY

[0004] The first object of the present application is to provide a preparation method of tirofiban-carrying super small prussian blue nanoparticles to overcome the deficiencies of the prior art. The tirofiban-carrying super small prussian blue nanoparticles prepared by the preparation method not only carry tirofiban, but also have catalase and superoxide dismutase mimetic nanoscale enzyme activity. Moreover, its size is up to nanoscale, which can be efficiently cleared through kidney excretion. In vitro experiments have confirmed its strong multi-species reactive oxygen species scavenging capacity. Through CREKA peptide modification, it has thrombus targeting property and can target the fibrin-fibronectin complex of thrombus. This targeting strategy can concentrate the therapeutic agent at the site of coronary thrombus, while reducing systemic exposure of the drug, thereby reducing the systemic adverse effects of the drug.

[0005] The above-mentioned objectives of the present invention are achieved through the following technical measures:

[0006] A method for preparing ultra-small Prussian blue nanoparticles carrying tirofiban is provided, comprising the following steps:

[0007] S1. Ultrasmall Prussian blue nanoparticles (USPB) were prepared by means of potassium ferricyanide, polyvinylpyrrolidone and hydrochloric acid-ethanol solution.

[0008] S2. Mix and stir tirofiban and the ultra-small Prussian blue nanoparticles USPB from S1 to obtain tirofiban-supported ultra-small Prussian blue (T-USPB).

[0009] S3. The tirofiban-loaded ultrasmall Prussian blue T-USPB of S2 is coupled with the CREKA peptide via carbodiimide-mediated coupling to obtain Prussian blue (T-USPB-C) of tirofiban-loaded with CREKA peptide modified with CREKA peptide.

[0010] Preferably, S1 above specifically involves: dissolving potassium ferricyanide and polyvinylpyrrolidone in hydrochloric acid-ethanol solution and reacting, then purifying by ultrafiltration and retaining the concentrate, and washing the concentrate with ultrapure water and centrifuging to obtain ultrasmall Prussian blue nanoparticles (USPB).

[0011] Preferably, S2 is performed by the following steps:

[0012] S2.1. Disperse the ultra-small Prussian blue nanoparticles (USPB) of S1 in ultrapure water A to obtain mixture A; dissolve tirofiban in ultrapure water B to obtain mixture B;

[0013] S2.2 Mix mixture A and mixture B together and stir to react, then centrifuge and wash the precipitate to obtain the supported tirofiban ultra-small Prussian blue (T-USPB).

[0014] Preferably, S3 is performed by the following steps:

[0015] S3.1 Disperse the loaded tirofiban ultra-small Prussian blue (T-USPB) obtained in S2 in MES buffer and proceed to S3.2;

[0016] S3.2, Add a crosslinking agent composed of EDC and NHS, then proceed to S3.3;

[0017] S3.3 Add CREKA peptide, stir the reaction, centrifuge, wash the precipitate to obtain CREKA peptide-modified tirofiban-loaded Prussian blue (T-USPB-C).

[0018] In S1, the weight of potassium ferricyanide: the weight of polyvinylpyrrolidone: the volume of hydrochloric acid-ethanol solution is (15-40) mg: (500-1000) mg: (5-100) mL.

[0019] Preferably, the above-mentioned hydrochloric acid-ethanol solution is a hydrochloric acid-75% ethanol solution and the concentration of hydrochloric acid is 0.01M.

[0020] In S2, the weight of the ultra-small Prussian blue nanoparticles USPB: the volume of ultrapure water A: the weight of tirofiban: the volume of ultrapure water B is (15-25) mg: (10-100) mL: (5-15) mg: (10-100) mL.

[0021] In S3, the weight of tirofiban-loaded ultra-small Prussian blue T-USPB: the volume of MES buffer: the weight of cross-linking agent: the weight of CREKA peptide is (5-15) mg: (5-50) mL: (20-40) mg: (5-20) mg.

[0022] In the crosslinking agent, the molar ratio of EDC to NHS is (0.1 to 10):1.

[0023] In S1, the weight of potassium ferricyanide: the weight of polyvinylpyrrolidone: the volume of hydrochloric acid-ethanol solution is 27.5 mg: 750 mg: 10 mL.

[0024] In S2, the ratio of USPB weight: ultrapure water A volume: tirofiban weight: ultrapure water B volume is 20 mg: 20 mL: 10 mg: 20 mL.

[0025] In S3, the weight of T-USPB: the volume of MES buffer: the weight of cross-linking agent: the weight of CREKA peptide is 10 mg: 10 mL: 32 mg: 10 mg.

[0026] In the crosslinking agent, the molar ratio of EDC to NHS is 1:1.

[0027] Preferably, S1 specifically involves: dissolving potassium ferricyanide and polyvinylpyrrolidone in hydrochloric acid-ethanol solution and reacting for 4 to 10 hours, then ultrafiltration under a molecular weight cutoff of 10 kDa, followed by centrifugation at 3000 g to 8000 g for 10 to 60 minutes to retain the concentrate, washing the concentrate with ultrapure water and centrifuging to obtain ultrasmall Prussian blue nanoparticles (USPB).

[0028] Preferably, S2.2 above specifically involves: mixing mixture A and mixture B and stirring at room temperature for 10-24 hours, then centrifuging and washing the precipitate to obtain the supported tirofiban ultra-small Prussian blue (T-USPB).

[0029] Preferably, step S3.2 specifically involves adding a crosslinking agent composed of EDC and NHS, activating it at room temperature for 10 to 60 minutes, and then proceeding to step S3.3.

[0030] Preferably, step S3.3 is as follows: CREKA peptide is added and stirred for 10-24 hours, the reactants are centrifuged at 8000g-12000g for 10-60 minutes, and the precipitate is washed with ultrapure water to obtain Prussian blue (T-USPB-C) modified with CREKA peptide and loaded with tirofiban.

[0031] Preferably, the nanoscale size of the aforementioned ultrasmall Prussian blue nanoparticles (USPB) is less than 10 nm.

[0032] Preferably, the MES buffer concentration is 0.1M and the pH value is 6.0.

[0033] The second objective of this invention is to overcome the shortcomings of existing technologies by providing ultra-small Prussian blue nanoparticles carrying tirofiban. These ultra-small Prussian blue nanoparticles carrying tirofiban not only carry tirofiban but also possess nanozyme-like activities mimicking catalase and superoxide dismutase due to the properties of Prussian blue. Furthermore, their nanoscale size allows for efficient clearance via renal excretion. In vitro experiments have demonstrated their powerful multi-species reactive oxygen species scavenging ability. Through CREKA peptide modification, they exhibit specificity, recognizing the abundant fibroin-fibronectin complex present in thrombi. This targeting strategy concentrates the therapeutic agent at the coronary thrombus site while reducing systemic exposure, thereby minimizing off-target effects.

[0034] A method for preparing ultra-small Prussian blue nanoparticles carrying tirofiban is provided, which is prepared by the above-described method for preparing ultra-small Prussian blue nanoparticles carrying tirofiban.

[0035] A third objective of this invention is to overcome the shortcomings of existing technologies by providing the use of tirofiban-loaded ultra-small Prussian blue nanoparticles in the preparation of drugs that reduce microvascular damage and microvascular injury (MIRI). These tirofiban-loaded ultra-small Prussian blue nanoparticles possess excellent reactive oxygen species scavenging capabilities, rapidly improving microvascular perfusion through antithrombotic effects, and subsequently exerting a sustained protective effect through antioxidant and anti-inflammatory mechanisms, effectively preventing microvascular damage and MIRI.

[0036] This invention provides the use of tirofiban-loaded ultra-small Prussian blue nanoparticles in the preparation of drugs that reduce MVO and MIRI, wherein the tirofiban-loaded ultra-small Prussian blue nanoparticles are prepared by the above-described method for preparing tirofiban-loaded ultra-small Prussian blue nanoparticles.

[0037] Preferably, the above-mentioned tirofiban-carrying ultrasmall Prussian blue nanoparticles (T-USPB-C) target the fibrin of thrombi.

[0038] Preferably, the above-mentioned tirofiban-carrying ultra-small Prussian blue nanoparticles (T-USPB-C) are delivered to the thrombus site via the CREKA peptide, thereby forming a high concentration of tirofiban locally.

[0039] Preferably, the Prussian blue (T-USPB-C) in the above-mentioned tirofiban-carrying ultra-small Prussian blue nanoparticles scavenges pathological reactive oxygen species by mimicking the activities of catalase and superoxide dismutase, thereby breaking the microthrombus formation mechanism caused by oxidative stress.

[0040] Preferably, the anti-inflammatory effect of Prussian blue in the above-mentioned tirofiban-carrying ultra-small Prussian blue nanoparticles (T-USPB-C) reduces the infiltration of inflammatory cells.

[0041] This invention discloses tirofiban-loaded ultra-small Prussian blue nanoparticles, their preparation method, and their use in the preparation of drugs that reduce MVO and MIRI. The preparation method of the tirofiban-loaded ultra-small Prussian blue nanoparticles comprises the following steps: S1, preparing ultra-small Prussian blue nanoparticles (USPB) using potassium ferricyanide, polyvinylpyrrolidone, and hydrochloric acid-ethanol solution; S2, mixing and stirring tirofiban and the USPB nanoparticles from S1 to obtain tirofiban-loaded ultra-small Prussian blue T-USPB; S3, coupling the T-USPB from S2 with a CREKA peptide via carbodiimide-mediated coupling to obtain CREKA peptide-modified tirofiban-loaded Prussian blue T-USPB-C, which is the tirofiban-loaded ultra-small Prussian blue nanoparticle. The present invention provides ultra-small Prussian blue nanoparticles carrying tirofiban, which possess nanozyme-like activities mimicking catalase and superoxide dismutase. Furthermore, their nanoscale size allows for efficient renal excretion, minimizing potential long-term toxicity. These ultra-small Prussian blue nanoparticles carrying tirofiban exhibit excellent reactive oxygen species scavenging capabilities, rapidly improving microvascular perfusion through antithrombotic effects, and subsequently providing sustained protection through antioxidant and anti-inflammatory mechanisms, effectively preventing microvascular damage and microvascular injury-related respiratory infections (MIRIs). Attached Figure Description

[0042] The invention will be further illustrated with reference to the accompanying drawings, but the contents of the drawings do not constitute any limitation on the invention.

[0043] Figure 1 This is a flowchart illustrating the preparation process of the tirofiban-loaded ultrasmall Prussian blue nanoparticles (T-USPB-C) of the present invention.

[0044] Figure 2 Characterization of T-USPB-C, where Figure 2 a is a transmission electron microscope image of USPB, T-USPB, and T-USPB-C; Figure 2 b represents the particle size distribution of USPB; Figure 2 c represents the particle size distribution of T-USPB; Figure 2 d represents the particle size distribution of T-USPB-C; Figure 2 e represents the release curve of tirofiban; Figure 2 f represents the USPB peroxidase activity as tested by the TMB and ABTS colorimetric reactions; Figure 2 g represents the scavenging efficiency of DPPH free radicals by different concentrations of USPB; Figure 2 h represents the scavenging efficiency of superoxide anions by different concentrations of USPB; Figure 2 i represents the scavenging efficiency of USPB against hydroxyl radicals. Figure 2 j represents the removal efficiency of hydrogen peroxide by different concentrations of USPB.

[0045] Figure 3 The in vitro antioxidant enzyme activity of T-USPB-C, of ​​which Figure 3 a) Live / dead images of human umbilical vein endothelial cells (HUVEC) and rat cardiomyocytes (H9C2) after culture in USPB, T-USPB, and T-USPB-C. Figure 3 b represents the survival rate of HUVEC cells after culturing with different concentrations of T-USPB-C nanoparticles; Figure 3 c represents the survival rate of H9C2 cells after being cultured with different concentrations of T-USPB-C nanoparticles; Figure 3 Image d shows the fluorescence staining image of T-USPB-C fluorescent nanoparticles entering H9C2 cardiomyocytes.

[0046] Figure 4 Fluorescence pattern for nanoparticles scavenging reactive oxygen species. Figure 4 a represents rat cardiomyocytes (H9C2). Figure 4 c shows the fluorescence of reactive oxygen species in human umbilical vein endothelial cells (HUVECs) in the normal control group, hypoxia-reoxygenation group, hypoxia-reoxygenation + tirofiban group, hypoxia-reoxygenation + T-USPB group, and hypoxia-reoxygenation + T-USPB-C group. Figure 4 Statistical chart where bc is 4a; Figure 4 A statistical chart where d represents 4c. Figure 4 a and Figure 4 The scale of d was 250 μm, n = 3, ns: no significant difference, ***P < 0.001.

[0047] Figure 5a represents HE staining of carotid artery thrombi treated with different nanoparticles, namely sham group, PBS group, tirofiban group, T-USPB group, and T-USPB-C group, with a scale of 200 μm; Figure 5 b represents the quantitative statistics of 5a; Figure 5 c is a representative image of DHE staining of the carotid artery, representing the effect of different nanoparticles on reactive oxygen species in the carotid artery, with a scale of 250 μm; Figure 5 Quantitative statistical analysis of d = 5c, and n = 3, **P < 0.01, ***P < 0.001.

[0048] Figure 6 a represents echocardiograms of different particle treatments 28 days later, B-mode and M-mode echocardiograms, respectively for the sham surgery group, PBS group, tirofiban group, T-USPB group, and T-USPB-C group. Figure 6 b represents the quantitative statistics of left ventricular shortening fraction (FS) from echocardiography; Figure 6 c represents the quantitative statistics of left ventricular ejection fraction (EF) from echocardiography; Figure 6 d shows cardiac TTC staining in different treatment groups; Figure 6 e is Figure 6 Quantitative statistics of myocardial infarction area of ​​d, with scale bar: 1 mm, n=3, ns: no significant difference, P<0.05, P<0.01, P<0.001.

[0049] Figure 7 To evaluate the effect of T-USPB-C on myocardial ischemia-reperfusion injury, Figure 7 a shows Masson and Sirius red staining images of the heart 28 days after T-USPB-C treatment, used to detect the degree of myocardial fibrosis. The scale bar is 1 mm. Figure 7 b represents the quantitative statistics of the fibrotic region of the left ventricular free wall; Figure 7 c is a DHE staining image of the degree of myocardial oxidation after treatment with different nanoparticles, and the scale bar is 100 μm; Figure 7 d represents the quantitative analysis of DHE staining fluorescence in the myocardium; Figure 7 e is a representative image of α-SMA immunofluorescence staining in the peripheral region 28 days after treatment, with a scale bar of 100 μm. Figure 7 f represents the quantitative analysis of myocardial α-SMA staining fluorescence, and n = 3, P < 0.01, *P < 0.001, ****P < 0.0001.

[0050] Figure 8 The inflammatory regulatory effect of T-USPB-C, among which Figure 8 a is an image of myocardial tissue treated with different nanoparticles and stained with HE; Figure 8b shows immunofluorescence staining images of macrophages in myocardial tissue after different nanoparticle treatments, where CD68 (red) represents total macrophages and iNOS (green) represents M1 macrophages; Figure 8 c represents the expression levels of the target protein and pro-inflammatory factors after different treatments; Figure 8 d represents the expression level of the target protein and the degree of apoptosis after different treatments.

[0051] Figure 9 The distribution maps of T-USPB and T-USPB-C in mouse in vivo imaging are shown, representing carotid artery thrombosis model, myocardial ischemia-reperfusion model, and carotid artery thrombosis + myocardial ischemia-reperfusion model, respectively, with a scale bar of 2 cm.

[0052] Figure 10 HE staining of major organs of mice after nanoparticle treatment, in sham-operated group and T-USPB-C group, with a scale bar of 200 μm. Detailed Implementation

[0053] The technical solution of the present invention will be further described in conjunction with the following embodiments.

[0054] Example 1

[0055] A method for preparing ultra-small Prussian blue nanoparticles carrying tirofiban, such as... Figure 1 The process is as follows:

[0056] S1. Ultrasmall Prussian blue nanoparticles (USPB) were prepared by potassium ferricyanide, polyvinylpyrrolidone and hydrochloric acid-ethanol solution.

[0057] S2. Mix and stir tirofiban and S1 ultra-small Prussian blue nanoparticles USPB to obtain tirofiban-supported ultra-small Prussian blue T-USPB.

[0058] S3. The tirofiban-loaded ultrasmall Prussian blue T-USPB from S2 is coupled to a CREKA peptide via carbodiimide-mediated coupling to obtain CREKA peptide-modified tirofiban-loaded Prussian blue T-USPB-C. This CREKA peptide-modified tirofiban-loaded Prussian blue T-USPB-C is the tirofiban-loaded ultrasmall Prussian blue nanoparticle. The ultrasmall Prussian blue nanoparticles (USPB) have a nanoscale size of less than 10 nm.

[0059] S1 specifically involves dissolving potassium ferricyanide and polyvinylpyrrolidone in a hydrochloric acid-ethanol solution and reacting them. Then, the solution is purified by ultrafiltration, and the concentrate is retained. The concentrate is washed with ultrapure water and centrifuged to obtain ultra-small Prussian blue nanoparticles (USPB).

[0060] S2 is performed by the following steps:

[0061] S2.1. Disperse the ultra-small Prussian blue nanoparticles USPB from S1 in ultrapure water A to obtain mixture A; dissolve tirofiban in ultrapure water B to obtain mixture B.

[0062] S2.2 Mix mixture A and mixture B together and stir to react, then centrifuge and wash the precipitate to obtain the supported tirofiban ultra-small Prussian blue T-USPB.

[0063] S3 is performed by the following steps:

[0064] S3.1 Disperse the tirofiban-loaded ultra-small Prussian blue T-USPB obtained in S2 in MES buffer and proceed to S3.2;

[0065] S3.2, Add a crosslinking agent composed of EDC and NHS, then proceed to S3.3;

[0066] S3.3 Add CREKA peptide, stir the reaction, centrifuge, wash the precipitate to obtain Prussian blue T-USPB-C modified with CREKA peptide and loaded with tirofiban.

[0067] It should be noted that tirofiban is a potent glycoprotein IIb / IIIa (GPⅡb / Ⅲa) receptor antagonist that effectively inhibits the final common pathway of platelet activation, adhesion, and aggregation. However, although tirofiban can reduce distal embolism and improve microvascular perfusion, its clinical application is severely limited by systemic administration-related bleeding complications. Prussian blue has an open coordination framework, an extremely high surface area to volume ratio, and significant adsorption capacity, thus achieving highly efficient drug loading. More importantly, its redox-active iron centers (Fe) in its crystal lattice... 3+ / Fe 2+ Prussian blue possesses various enzyme-mimicking activities, including catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD)-like functions, thus enabling it to effectively scavenge various reactive oxygen species (hydrogen peroxide, O2, etc.). 2- ,

[0068] · It generates molecular oxygen (OH) to alleviate tissue hypoxia, while also exhibiting anti-inflammatory and anti-apoptotic effects.

[0069] This invention utilizes an innovative synthetic method employing a mixed ethanol / water solvent and polyvinylpyrrolidone as a capping agent to develop ultra-small Prussian blue (USPB) nanoparticles with a diameter significantly smaller than that of conventional Prussian blue. Experiments have demonstrated that these USPBs exhibit superior antioxidant capabilities and, due to their nanoscale size (<10 nm), can be efficiently eliminated through renal excretion, thereby minimizing potential long-term toxicity issues. To further enhance therapeutic targeting, this invention employs the CREKA peptide (Cys-Arg-Glu-Lys-Ala), which specifically recognizes the fibronectin-fibronectin complex, which is abundant in pathological thrombi but almost undetectable in normal circulation.

[0070] In S1, the weight of potassium ferricyanide: the weight of polyvinylpyrrolidone: the volume of hydrochloric acid-ethanol solution is (15-40) mg: (500-1000) mg: (5-100) mL.

[0071] The hydrochloric acid-ethanol solution is a hydrochloric acid-75% ethanol solution with a hydrochloric acid concentration of 0.01 M. The 75% ethanol solution has a volume fraction of 75%.

[0072] In S2, the weight of ultra-small Prussian blue nanoparticles USPB: the volume of ultrapure water A: the weight of tirofiban: the volume of ultrapure water B is (15-25) mg: (10-100) mL: (5-15) mg: (10-100) mL.

[0073] In S3, the weight ratio of tirofiban-loaded ultra-small Prussian blue T-USPB to MES buffer volume to crosslinking agent weight to CREKA peptide weight was (5–15) mg: (5–50) mL: (20–40) mg: (5–20) mg. The MES buffer concentration was 0.1 M and the pH was 6.0.

[0074] In the crosslinking agent, the molar ratio of EDC to NHS is (0.1–10):1.

[0075] S1 is specifically as follows: potassium ferricyanide and polyvinylpyrrolidone are dissolved in hydrochloric acid-ethanol solution and reacted for 4h to 10h. Then, ultrafiltration is performed under a molecular weight cutoff of 10kDa. The solution is then centrifuged at 3000g to 8000g for 10min to 60min and the concentrate is retained. The concentrate is washed with ultrapure water and centrifuged to obtain ultra-small Prussian blue nanoparticles (USPB).

[0076] S2.2 Specifically, mixture A and mixture B are mixed and stirred at room temperature for 10-24 hours, then centrifuged and the precipitate is washed to obtain the supported tirofiban ultra-small Prussian blue T-USPB.

[0077] S3.2 specifically involves adding a crosslinking agent composed of EDC and NHS, activating it at room temperature for 10 to 60 minutes, and then proceeding to S3.3.

[0078] S3.3 Specifically, CREKA peptide is added and stirred for 10-24 hours. The reactants are centrifuged at 8000g-12000g for 10-60 minutes. The precipitate is then washed with ultrapure water to obtain Prussian blue T-USPB-C modified with CREKA peptide and loaded with tirofiban.

[0079] The method for preparing ultra-small Prussian blue nanoparticles carrying tirofiban describes a process that produces nanoparticles capable of carrying tirofiban and exhibiting catalase and superoxide dismutase-mimicking nanozyme activities. Furthermore, their nanoscale size allows for efficient renal excretion, minimizing potential long-term toxicity. These ultra-small Prussian blue nanoparticles also demonstrate excellent reactive oxygen species (ROS) scavenging capabilities. They rapidly improve microvascular perfusion through antithrombotic effects, followed by sustained protective effects through antioxidant and anti-inflammatory mechanisms, effectively preventing microvascular damage and microvascular injury-related respiratory infections (MIRIs).

[0080] Example 2

[0081] A method for preparing ultrasmall Prussian blue nanoparticles carrying tirofiban is provided. Other features are the same as in Example 1, but the method also has the following features: S1 is as follows: potassium ferricyanide and polyvinylpyrrolidone are dissolved in hydrochloric acid-ethanol solution and reacted for 10 h. Then, ultrafiltration is performed under a molecular weight cutoff of 10 kDa. The solution is then centrifuged at 3000 g for 60 min and the concentrate is retained. The concentrate is washed with ultrapure water and centrifuged to obtain ultrasmall Prussian blue nanoparticles USPB.

[0082] S2.2 Specifically, mixture A and mixture B are mixed and stirred at room temperature for 10 hours, then centrifuged and the precipitate is washed to obtain the supported tirofiban ultra-small Prussian blue T-USPB.

[0083] S3.2 specifically involves adding a crosslinking agent composed of EDC and NHS, activating it at room temperature for 10 minutes, and then proceeding to S3.3.

[0084] S3.3 Specifically, CREKA peptide was added and stirred for 10 h. The reactants were centrifuged at 8000 g for 60 min, and then the precipitate was washed with ultrapure water to obtain Prussian blue T-USPB-C modified with CREKA peptide and loaded with tirofiban.

[0085] In S1, the weight of potassium ferricyanide: the weight of polyvinylpyrrolidone: the volume of hydrochloric acid-ethanol solution is 15 mg: 500 mg: 100 mL.

[0086] In S2, the weight of ultrasmall Prussian blue nanoparticles USPB: the volume of ultrapure water A: the weight of tirofiban: the volume of ultrapure water B is 15 mg: 10 mL: 15 mg: 100 mL.

[0087] In S3, the weight of tirofiban-loaded ultra-small Prussian blue T-USPB: volume of MES buffer: weight of cross-linking agent: weight of CREKA peptide was 15 mg: 50 mL: 20 mg: 5 mg.

[0088] In the crosslinking agent, the molar ratio of EDC to NHS is 0.1:1.

[0089] Example 3

[0090] A method for preparing ultrasmall Prussian blue nanoparticles carrying tirofiban is provided. Other features are the same as in Example 1, but the method also has the following features: S1 is as follows: potassium ferricyanide and polyvinylpyrrolidone are dissolved in hydrochloric acid-ethanol solution and reacted for 4 hours. Then, ultrafiltration is performed under a molecular weight cutoff of 10 kDa. The solution is then centrifuged at 8000 g for 10 min and the concentrate is retained. The concentrate is washed with ultrapure water and centrifuged to obtain ultrasmall Prussian blue nanoparticles USPB.

[0091] S2.2 Specifically, mixture A and mixture B are mixed and stirred at room temperature for 24 hours, then centrifuged and the precipitate is washed to obtain the supported tirofiban ultra-small Prussian blue T-USPB.

[0092] S3.2 specifically involves adding a crosslinking agent composed of EDC and NHS, activating it at room temperature for 60 minutes, and then proceeding to S3.3.

[0093] S3.3 Specifically, CREKA peptide was added and stirred for 24 h. The reactants were centrifuged at 12000 g for 10 min, and then the precipitate was washed with ultrapure water to obtain Prussian blue T-USPB-C modified with CREKA peptide and loaded with tirofiban.

[0094] In S1, the weight of potassium ferricyanide: the weight of polyvinylpyrrolidone: the volume of hydrochloric acid-ethanol solution is 40 mg: 1000 mg: 100 mL.

[0095] In S2, the weight of ultra-small Prussian blue nanoparticles USPB: the volume of ultrapure water A: the weight of tirofiban: the volume of ultrapure water B is 25 mg: 100 mL: 5 mg: 10 mL.

[0096] In S3, the weight of tirofiban-loaded ultra-small Prussian blue T-USPB: volume of MES buffer: weight of cross-linking agent: weight of CREKA peptide was 5 mg: 5 mL: 40 mg: 20 mg.

[0097] In the crosslinking agent, the molar ratio of EDC to NHS is 10:1.

[0098] Example 4

[0099] A method for preparing ultrasmall Prussian blue nanoparticles carrying tirofiban is provided, which is the same as in Example 1 in other aspects, and also has the following features: S1 specifically involves dissolving potassium ferricyanide and polyvinylpyrrolidone in hydrochloric acid-ethanol solution and reacting for 7 hours, then ultrafiltration under a molecular weight cutoff of 10 kDa, followed by centrifugation at 5000 g for 30 minutes to retain the concentrate, washing the concentrate with ultrapure water and centrifuging again to obtain ultrasmall Prussian blue nanoparticles USPB.

[0100] S2.2 Specifically, mixture A and mixture B are mixed and stirred at room temperature for 15 hours, then centrifuged, and the precipitate is washed to obtain the supported tirofiban ultra-small Prussian blue T-USPB.

[0101] S3.2 specifically involves adding a crosslinking agent composed of EDC and NHS, activating it at room temperature for 30 minutes, and then proceeding to S3.3.

[0102] S3.3 Specifically, CREKA peptide was added and stirred for 16 h. The reactants were centrifuged at 10000 g for 30 min, and then the precipitate was washed with ultrapure water to obtain Prussian blue T-USPB-C modified with CREKA peptide and loaded with tirofiban.

[0103] In S1, the weight of potassium ferricyanide: the weight of polyvinylpyrrolidone: the volume of hydrochloric acid-ethanol solution is 27.5 mg: 750 mg: 10 mL.

[0104] In S2, the weight of ultrasmall Prussian blue nanoparticles USPB: the volume of ultrapure water A: the weight of tirofiban: the volume of ultrapure water B is 20 mg: 20 mL: 10 mg: 20 mL.

[0105] In S3, the weight of tirofiban-loaded ultra-small Prussian blue T-USPB: volume of MES buffer: weight of cross-linking agent: weight of CREKA peptide was 10 mg: 10 mL: 32 mg: 10 mg.

[0106] In the crosslinking agent, the molar ratio of EDC to NHS is 1:1.

[0107] Experimental Example

[0108] 1. Model building method

[0109] 1.1 Methods for establishing a hypoxia-reoxygenation cell model

[0110] A hypoxia-reoxygenation model was established using H9C2 and HUVEC cells to simulate oxidative stress damage in cardiomyocytes and vascular endothelial cells during reperfusion. Cells were cultured for 48 hours in a high-glucose medium containing 10% fetal bovine serum and antibiotics. When cell confluence reached approximately 80%, the medium was replaced with an equal volume of PBS. Cells were then subjected to hypoxia for 3 hours in a tri-gas incubator with 1% oxygen. After hypoxia, the PBS was replaced with pre-prepared culture media containing different drugs (tirofiban, T-USPB, and T-USPB-C), and the cells were cultured for another 6 hours in a cell culture incubator at 37°C and 5% CO2.

[0111] 1.2 Methods for establishing a mouse model of myocardial ischemia-reperfusion injury (MIRI)

[0112] An I / R model was established using male C57BL / 6 mice (18-22g). Mice were anesthetized with sodium pentobarbital (1.5mg / 20g) and ventilated using a DW-3000B rodent ventilator (Beijing Xinxing, China). The chest skin was disinfected, and an incision was made in the fourth intercostal space to fully expose the heart. The left anterior descending artery (LAD) was ligated with 8-0 silk suture for 40 minutes, then the ligation was released to restore blood flow to the LAD. The thoracic cavity was then closed, the skin was sutured, and the area was disinfected. For three consecutive days after the ligation was released, mice were administered 200μL of tirofiban, T-USPB, and T-USPB-C via tail vein injection. The untreated group received 200μL of PBS. The sham-operated group underwent the same procedure but without LAD ligation. Mice were fed normally after waking from anesthesia.

[0113] 1.3 Methods for establishing a mouse carotid artery thrombosis model

[0114] A carotid artery thrombosis model was established using male C57BL / 6 mice (18-22g). Mice were anesthetized with sodium pentobarbital (1.5mg / 20g), and the neck skin was disinfected. An incision was made along the midline of the neck, and muscles and tissues were bluntly dissected to expose the right carotid artery adjacent to the trachea. A piece of paper (5mm×5mm) was placed under the carotid artery to prevent damage to other tissues during the modeling process. The carotid artery was immersed in 10% FeCl3 solution for 3 minutes. After modeling, the carotid artery was rinsed twice with PBS. Within 5 minutes after modeling, mice were injected via the tail vein with an insulin syringe containing 90μg / kg tirofiban, T-USPB, and T-USPB-C in a volume of 200μL. The untreated group received 200μL of PBS. The sham-operated group underwent only a neck incision and no further treatment. After the mice awoke from anesthesia, they were fed as usual.

[0115] All laboratory animal procedures were approved by the Laboratory Animal Ethics Committee of Zhujiang Hospital, Southern Medical University (Approval No. LAEC-2025-008). Animal use complied with the guidelines issued by the National Institutes of Health (NIH).

[0116] Laboratory Animal Care and Use Guidelines (NIH Publication, 8th Edition, 2011).

[0117] 2. Characterization methods for USPB, T-USPB, and T-USPB-C

[0118] This invention systematically characterized the successful preparation of USPB using complementary analytical techniques. Transmission electron microscopy (TEM) analysis was performed using a JEM-2100F instrument to determine primary particle size and morphology, while dynamic light scattering (DLS) measurements were performed to assess the hydrodynamic diameter and size distribution in the aqueous suspension.

[0119] Transmission electron microscopy (TEM) is used to observe the microstructure of nanoparticles, while dynamic light scattering (DLS) is used to determine the hydrodynamic dimensions of the USPB. Figure 2 a.

[0120] TEM observation showed that the nanoparticles exhibited a uniform spherical structure, with T-USPB-C having a larger size than both USPB and T-USPB. DLS measurements determined the average hydrodynamic dimensions of USPB, T-USPB, and T-USPB-C to be approximately 10 nm, 12 nm, and 15 nm, respectively. Figure 2 b- Figure 2 d.

[0121] 3. Release kinetics of tirofiban in T-USPB-C nanoparticles

[0122] Tirofiban was dissolved in ultrapure water and serially diluted to prepare aqueous solutions of different concentrations. Using ultrapure water as a blank reference, the absorbance of each tirofiban solution was measured at 227 nm using an ultra-micro UV-Vis spectrophotometer. The measured absorbance values ​​were then plotted against the corresponding concentrations using GraphPad software to generate a standard curve. The drug release curve of tirofiban from T-USPB-C nanoparticles was then studied using dialysis. Specifically, the nanoparticles were dispersed in PBS and placed in a dialysis bag with a molecular weight cutoff of 10 kDa. The dialysis bag was then immersed in 50 mL of PBS maintained at 37 °C with continuous stirring. At predetermined time points, 10 μL aliquots were collected from the external PBS solution, and the absorbance was measured at 227 nm using an ultra-micro UV-Vis spectrophotometer to analyze the tirofiban content. After each sampling, an equal volume of fresh PBS was immediately added to maintain a constant total volume. The absorbance values ​​were plotted against time using Graph Pad Prism software to generate the tirofiban cumulative release curve.Figure 2 e.

[0123] 4. In vitro enzyme activity assay method for ultra-small Prussian blue nanoparticles

[0124] The enzyme activity of these ultrasmall Prussian blue nanoparticles was systematically evaluated using three parallel enzyme simulation assays based on commercial kits.

[0125] For the activity assay of POD-like enzymes, the following results were obtained. Figure 2 f. The specific test method is as follows: First, weigh 20 mg of TMB and dissolve it in 50 mL of acetate buffer with a pH of 4.5. Heat the solution in a water bath to 40°C until the TMB is completely dissolved, thus preparing a TMB solution with a concentration of 0.4 mg / mL. Next, take 10 μL of 10 M H2O2 and add it to 50 mL of PBS buffer, then dilute it to a 2 mM H2O2 solution by vortex mixing. Subsequently, weigh a certain amount of USPB and add it to PBS buffer, and sonicate for 15 minutes to prepare suspensions with concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, and 30 μg / mL, respectively. Transfer these suspensions sequentially to disposable quartz cuvettes, add suspensions of different concentrations of ultrafine Prussian blue nanoparticles, and observe the color change of the solution after standing for 1 minute. Then, take 10 μL of the mixture and measure its absorbance in the 500–800 nm wavelength range using an ultra-micro UV-Vis spectrophotometer.

[0126] DPPH scavenging capacity was determined, and the results were obtained. Figure 2 g. Specific testing method: Accurately weigh DPPH powder and prepare a 0.1 mmol / L stock solution with anhydrous ethanol. Dilute to a 0.02 mmol / L working solution before the experiment (prepare fresh in the dark); disperse ultra-small Prussian blue nanoparticles in PBS (pH 7.4) and sonicate for 5–10 min to prevent aggregation, and prepare a series of concentration gradient solutions of 0.01–1.0 mg / mL. Simultaneously, set up a vitamin C (Vc) positive control and a blank control group (PBS only) with the same concentration gradient; perform high-throughput detection using a 96-well plate. Add 50 μL of sample solution to both the experimental group and the positive control group. 50 μL of DPPH working solution (total system 200 μL) was used. The DPPH control group was replaced with 50 μL of anhydrous ethanol instead of the sample solution, and the sample control group was replaced with 50 μL of anhydrous ethanol instead of the DPPH working solution. Each group was set up with 3 replicates. After vortexing, the solution was incubated at room temperature in the dark for 30 min. The absorbance of each well was measured at a wavelength of 517 nm using an ELISA reader (denoted as A1, A2, A0, and A sample, respectively). The clearance efficiency was calculated according to the formula: clearance rate (%) = [1 - (A1 - A sample) / A0] × 100%, where A1 - A sample was used to deduct the interference of the nanozyme's own absorbance, and A0 was the initial absorbance of DPPH.

[0127] When its SOD-like activity was detected, the following was obtained: Figure 2 h. The specific testing method was as follows: quantitative analysis was performed using a WST-8-based assay. The specific procedure was as follows: a predetermined amount of ultra-small Prussian blue nanoparticles were dispersed in PBS buffer by ultrasonic treatment (15 minutes) to create a concentration gradient (5, 10, 20, and 30 μg / mL). These nanoparticle suspensions were then aliquoted into 96-well plates, along with corresponding control samples (including sample, control group, blank 1, and blank 2). After incubation at 37°C for 30 minutes, absorbance was measured at 450 nm (A_sample, A_control, A_blank1, and A_blank2, respectively). The superoxide anion scavenging capacity was calculated using the formula: inhibition rate (%) = [(ΔA_blank - ΔA_sample) / ΔA_blank] × 100%, where ΔA_sample represents the absorbance difference between the sample and control group, and ΔA_blank represents the absorbance difference between blank 1 and blank 2. This multi-concentration analysis method (repeated three times for each concentration point) performed under standardized conditions (37±0.5℃) can reliably characterize the dose-dependent SOD simulation activity of ultrasmall Prussian blue nanoparticles, and its inhibition rate can directly reflect its ability to scavenge reactive oxygen species.

[0128] The ability to scavenge hydroxyl radicals was detected and obtained. Figure 2 i. The specific testing method is as follows: Terephthalic acid (TA) is accurately weighed, dissolved with 0.1 mol / L NaOH solution, and then diluted with PBS (pH 7.4) to prepare a 5 mmol / L TA working solution; 5 mmol / L FFeSO4 solution and 10 mmol / L H2O2 solution are prepared separately (freshly prepared for use); ultra-small Prussian blue nanoparticles are dispersed in PBS and sonicated for 5–10 min to prevent aggregation, preparing a series of concentration gradient solutions of 0.05–2.0 mg / mL; simultaneously, a vitamin C (Vc) positive control and a blank control group (PBS only) are set up; 20 μL of FFeSO4 solution, 20 μL of TA working solution, and 20 μL of nanozyme sample solution are added sequentially to the reaction system (total volume 100 μL) (the positive control group is replaced with Vc solution, and the blank control group is replaced with PBS); finally, 40 μL of H2O2 solution is added to initiate the Fenton reaction (Fe... 2+ +H₂O₂→Fe 3+ +·OH+OH -Each group was configured with 3 duplicate wells. After vortex mixing, the mixture was incubated at 37°C in the dark for 60 min. The fluorescence intensity was measured using a fluorescence spectrophotometer (excitation wavelength 315 nm, emission wavelength 425 nm). The fluorescence values ​​of the experimental group (F1), positive control group (F2), ·OH control group (F0, with PBS instead of nanozyme), sample control group (F sample, with PBS instead of H2O2), and blank control group (F blank, PBS only) were recorded. The scavenging efficiency was calculated using the formula: scavenging rate (%) = [1 - (F1 - F sample) / (F0 - F blank)] × 100%. (F1 - F sample is calculated after deducting the fluorescence interference of the nanozyme itself, F0 - F blank is calculated as follows). · The maximum fluorescence value produced by the reaction of OH with TA was used to plot the dose-response curve with nanozyme concentration on the x-axis and clearance rate on the y-axis.

[0129] For the activity assay of CAT-like enzymes, the following results were obtained. Figure 2 j. The specific testing method is as follows: First, a certain amount of ultra-small Prussian blue nanoparticles were weighed and added to PBS buffer. The mixture was then sonicated for 15 minutes to prepare suspensions with concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, and 30 μg / mL. The spectrophotometer was preheated for at least 30 minutes, and the measurement wavelength was set to 240 nm. Zero-point calibration of the instrument was performed using distilled water. Before measurement, the CAT enzyme activity assay working solution was incubated in a 37°C water bath for 10 minutes. Then, 1 mL of the working solution was placed in a disposable quartz cuvette, and 35 μL of sample was added. The mixture was mixed for 5 seconds. After incubation at room temperature for 1 minute, the absorbance was measured at 240 nm. Under the catalytic action of the nanozyme, H2O2 decomposes into oxygen and water. Therefore, the characteristic absorption peak of hydrogen peroxide at 240 nm gradually weakens over time. This phenomenon indicates that the nanozyme has a catalytic effect similar to that of the CAT enzyme.

[0130] 5. Cell viability assay methods

[0131] A hypoxia-reoxygenation cell model was used, with H9C2 cells derived from embryonic rat hearts cultured in 100 cm² cell culture flasks. Cells were cultured in DMEM (Gibco, USA) supplemented with 10% FBS (Gibco, USA) and 1% penicillin / streptomycin at 37°C and 5% CO₂. The culture medium was changed every 2 days. Cells were passaged when confluence reached 80%. HUVECs derived from human umbilical vein endothelial cells were cultured under the same conditions, with passage and culture time adjusted according to cell growth status.

[0132] To assess the biocompatibility of the nanoparticles, a CCK-8 assay was performed to analyze their effect on cell growth. H9C2 cells and HUVECs cells were cultured at 4 × 10⁻⁶ cells / year. 4Cells were seeded at a density of 100 cells / well in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours. After removing the culture medium, the cells were treated with culture medium containing different concentrations of T-USPB-C (12.5, 25, 50, 100, and 200 μg / mL) for 48 hours, with five replicates for each concentration. The culture medium was then discarded, and 10% CCK-8 solution (Yeasen, China) was added to each well, followed by further incubation at 37°C with 5% CO2 for 2 hours. Subsequently, the absorbance of the culture medium was measured using a Varioskan LUX multi-mode microplate reader (Thermo Fisher, USA). Figure 3 a-3c.

[0133] 6. Detection methods for reactive oxygen species in cells

[0134] To determine whether nanoparticles affect the accumulation of reactive oxygen species (ROS), DCFH-DA staining was used to observe ROS accumulation in H9C2 and HUVEC cells after hypoxia-reoxygenation. Cells were cultured in 35 mm culture dishes as shown above. This assay used a hypoxia-reoxygenation model established with H9C2 and HUVEC cells, which were then incubated with tirofiban, T-USPB, and T-USPB-C for 6 hours, respectively. After discarding the culture medium and washing three times, 1 mL of DCFH-DA working solution prepared at a 1:1000 ratio was added, and staining was performed in the dark for 30 minutes. Then, the culture medium was discarded, and after washing three times, Hoechst working solution prepared at a 1:1000 ratio was added to the culture medium, and staining was performed for 15 minutes. Finally, the culture medium was discarded, replaced with DMEM, and observed under a fluorescence microscope. The relative fluorescence intensity was quantified using ImageJ software, expressed as the DCFH-DA fluorescence intensity per cell, as shown below. Figure 4 .

[0135] 7. Evaluation method for antithrombotic effect in mice

[0136] A mouse carotid artery thrombosis model was used. On day 1 post-treatment, mice were anesthetized and sacrificed. The carotid artery was removed, fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned. Hematoxylin and eosin (H&E) staining was performed to assess the improvement of carotid artery thrombosis. Sections were dewaxed, rehydrated, stained with nuclear and cytoplasmic reticulum, dehydrated, and mounted before observation. ImageJ was used to assess the thrombus area; thrombus size was expressed as the ratio of thrombus area to the total vascular area. Figure 5 a-5b.

[0137] 8. Detection methods for reactive oxygen species in the carotid artery and myocardium of mice.

[0138] A mouse model of myocardial ischemia-reperfusion injury (MIRI) was used. On day 1 post-treatment, mice were anesthetized and sacrificed, and the carotid artery was removed. On day 3 post-treatment, mice were anesthetized and sacrificed, and the heart was removed. The area from the ligation site to the apex of the heart was excised. The tissue was embedded in OCT, and frozen sections were prepared at the injury site. The sections were washed three times with PBS, and DHE working solution prepared at a ratio of 1:1000 was added to the dried sections for staining. 60 μL was used for each heart tissue section, and 10 μL was used for each vascular tissue section. The sections were incubated at 37°C in the dark for 40 minutes. After washing three times with PBS and drying, the nuclei were stained with Hoechst working solution for 15 minutes. The sections were then washed three times with PBS for 5 minutes each time. After drying, the sections were mounted and observed under a fluorescence microscope. The relative fluorescence intensity was quantified using ImageJ software and expressed as the DHE fluorescence intensity per cell to obtain the reactive oxygen species (ROS) detection of the carotid artery. Figure 5 c-5d), cardiac reactive oxygen species detection ( Figure 7 e).

[0139] 9. Methods for assessing cardiac function in mice

[0140] After 28 days of treatment with different nanoparticles in mice undergoing ischemia-reperfusion injury, we assessed changes in cardiac function by echocardiography. Echocardiograms of the mice were acquired using an ultrasound probe in both M-mode and B-mode. Figure 6 a, Figure 6 b and Figure 6 c. After obtaining echocardiographic images, during data analysis, the value of each echocardiographic parameter is calculated as the average of three consecutive cardiac cycles. The calculation formula is as follows:

[0141] Ejection fraction EF = (Left ventricular end-diastolic volume LVEDV - Left ventricular end-systolic volume LVESV) / ​​LVEDV x 100%.

[0142] Short axis shortening rate FS = (left ventricular end-diastolic diameter LVEDd - left ventricular end-systolic diameter LVEDs) / LVEDd x 100%.

[0143] 10. Assessment of infarct area in mouse MIRI model

[0144] A mouse model of myocardial ischemia-reperfusion injury (MIRI) was used. On day 3 post-treatment, mice were anesthetized and sacrificed, and the heart was removed. The infarct area was assessed by TTC staining. After removal, the trimmed heart tissue was placed along its long axis on a 60 mm culture dish lid and rapidly frozen at -80°C for 10 minutes for sectioning. The fully frozen heart was sectioned along its long axis into 4-5 slices. Each heart was immersed in 2 mL of 1% TTC solution and incubated at 37°C in the dark for 10 minutes, with the staining time adjusted according to the staining results. After staining, one slice of heart tissue was fixed between two coverslips and then placed in 4% paraformaldehyde for 12 hours before photography. The infarct area was analyzed using ImageJ, and the infarct area was expressed as the ratio of the infarct area to the heart area. Figure 6 d and Figure 6 e.

[0145] 11. Assessment of myocardial fibrosis and angiogenesis in mice

[0146] The mouse model of myocardial ischemia-reperfusion injury (MIRI) was used. On day 28 post-treatment, mice were sacrificed, and the perfused and harvested hearts were sectioned in paraffin and then stained with Masson's trichrome. Figure 7 a) and Sirius red staining ( Figure 7 e) To assess myocardial fibrosis. ImageJ was used to measure scar area and left ventricular free wall area. The degree of fibrosis was expressed as the ratio of scar area to left ventricular free wall area. Figure 7 b). Paraffin sections were also used for immunofluorescence detection of α-SMA to assess angiogenesis. Figure 7 f).

[0147] 12. In vivo inflammation evaluation in mouse MIRI model

[0148] A mouse model of myocardial ischemia-reperfusion injury (MIRI) was used. Mice were anesthetized and sacrificed on day 3 post-treatment. Hearts were rapidly removed after PBS perfusion, then fixed, dehydrated, paraffin-embedded, and sectioned. Hematoxylin and eosin (H&E) staining was performed to assess inflammatory infiltration in the mouse hearts. Figure 8 a). Immunofluorescence detection was performed simultaneously. After dewaxing and rehydration of paraffin sections, antigen retrieval was performed, followed by permeabilization with 0.5% Triton for 15 minutes and blocking for 30 minutes. Primary antibody (macrophage marker CD68, iNOS) was incubated overnight at 4°C. The next day, after washing three times with PBST, the sections were incubated with fluorescent secondary antibody for 1 hour at room temperature in the dark. After washing three times with PBST again, the nuclei were stained with DAPI, and the sections were mounted with anti-fluorescence quenching mounting medium. Macrophage polarization was observed and evaluated using confocal microscopy. Figure 8 b).

[0149] 13. Western Blot

[0150] Heart tissue: On day 3 post-treatment, mice were sacrificed, and the hearts were perfused and removed. Myocardial tissue samples were trimmed from the damaged area, cut into small pieces, and placed in enzyme-free EP tubes. A grinding bead and 0.5 mL of RIPA lysis buffer containing 1% protease inhibitor and 1% phosphatase inhibitor were added for low-temperature grinding. The grinding bead was then removed, and the tissue was lysed by sonication, followed by centrifugation at 12,000 rpm for 15 minutes. The supernatant obtained was the tissue protein. Subsequent steps were the same as above to obtain… Figure 8 c and 8d.

[0151] 14. Small animal live imaging

[0152] Fluorescent probes were loaded onto nanoparticles (T-USPB, T-USPB-C) to prepare imaging agents. The loading process was performed under sterile conditions, with the mixture incubated at 37°C with gentle shaking for 2 hours to ensure uniform probe binding. After preparation, the imaging agent was centrifuged at 8000 rpm for 10 minutes to remove unbound fluorescent probes, and its concentration was adjusted to 0.5 mg / mL using phosphate-buffered saline (PBS) for later use.

[0153] Before the experiment, Kunming mice (6-8 weeks old, male, weighing 20-25g) were acclimatized to the laboratory environment for one week, with free access to food and water and a 12-hour light-dark cycle. For fluorescence imaging, the mice needed to be fasted overnight (approximately 12 hours) to reduce autofluorescence caused by food residue, while still allowing free access to water. On the day of the experiment, the mice were anesthetized with 2% isoflurane (oxygen flow rate: 1L / min) using an anesthesia machine, and their body temperature was maintained at 37±0.5℃ throughout the process to ensure physiological stability. The imaging agent was injected via the tail vein using a 30G needle, with an injection volume of 100μL per mouse. The injection was performed slowly, lasting 10-15 seconds to avoid vascular damage, and after injection, the injection site was gently pressed with sterile gauze for a few seconds to prevent drug leakage.

[0154] After allowing the imaging agent to circulate in vivo for 4 hours to ensure adequate targeting and accumulation, anesthetized mice were placed prone on the imaging platform of the in vivo imaging system. Imaging parameters were set as follows: excitation wavelength matched to the fluorescent probe (650 nm), emission wavelength (670 nm), exposure time 10 seconds, and field of view covering the entire body. Images of the target areas (neck, aorta, and heart) were acquired, with three duplicate images taken from each mouse to ensure data reliability. Figure 9 .

[0155] 15. Biocompatibility and safety testing

[0156] On day 3 after continuous intravenous treatment with T-USPB-C, mice were anesthetized and sacrificed. Other vital organs, such as the liver, spleen, lungs, and kidneys, were removed along with the heart. These organs were then fixed, dehydrated, embedded in paraffin, and sectioned. The paraffin sections were subsequently stained with hematoxylin and eosin (H&E) for morphological analysis. Figure 10 .

[0157] 16. Statistical Analysis

[0158] All experimental data are expressed as mean ± standard error of mean (Mean ± SEM). Data were analyzed using Prism 9 and SPSS 20.0 software, and statistical graphs were created using GraphPad Prism 8.0 software. The t-test was used for comparisons between two groups. ANOVA was used for comparisons involving more than two independent samples. For the analysis of repeated measures data, two-way repeated measures (RM) ANOVA was used, followed by a Bonferroni post-hoc test. A p-value < 0.05 was considered statistically significant.

[0159] Analysis example

[0160] 1. Morphological and dimensional characterization of T-USPB-C

[0161] Figure 2 a. Transmission electron microscopy (TEM) observation showed that the nanoparticles had a uniform spherical structure, and the size of T-USPB-C was larger than that of USPB and T-USPB.

[0162] like Figure 2 b- Figure 2 As can be seen, the average hydrodynamic dimensions of USPB, T-USPB and T-USPB-C determined by dynamic light scattering (DLS) are approximately 10 nm, 12 nm and 15 nm, respectively.

[0163] pass Figure 2 a- Figure 2 d indicates that the nanoparticles of the present invention are uniformly distributed and well dispersed.

[0164] 2. Release characterization of tirofiban in T-USPB-C

[0165] Figure 2 e- Figure 2 f. The relationship between tirofiban concentration and T-USPB-C nanoparticle solution was investigated by measuring the absorbance of T-USPB-C nanoparticle solutions with different tirofiban concentrations. The figure shows that the absorbance increases with increasing tirofiban concentration. The standard curve for tirofiban is expressed as: Y = 0.009933X + 0.008562(R 2=0.9987). This standard curve allows for the quantification of tirofiban concentration by absorbance measurement.

[0166] The in vitro release kinetics of tirofiban in T-USPB-C nanoparticles were evaluated using dialysis. A slow and sustained release pattern of tirofiban was observed over time, with a relatively linear release curve within one week and a cumulative release of 80% within 10 days. This slow and sustained release characteristic helps prolong the residence time of nanoparticles at the thrombus site, thereby extending the duration of drug action. This is significant for exerting antithrombotic effects and mitigating myocardial ischemia-reperfusion injury (MIRI).

[0167] 3. In vitro antioxidant enzyme activity of T-USPB-C

[0168] like Figure 2 As shown in f, the peroxidase activity of USPB was evaluated using the commonly used natural peroxidase (POD) substrates TMB and ABTS. When TMB was used as the substrate, the Km value of USPB was higher than that of ABTS, indicating that USPB had a stronger affinity for TMB than ABTS. This shows that USPB exhibits weak POD activity under neutral conditions. It shows almost no activity in alkaline environments, but exhibits POD activity in acidic environments. This also suggests that USPB may perform exceptionally well in slightly acidic wound microenvironments.

[0169] The antioxidant enzyme activity of Prussian blue increases with increasing concentration. For example... Figure 2 g- Figure 2 As shown in Figure j, at a concentration of 30 μg / mL, Prussian blue exhibits a scavenging rate of approximately 84% for ·OH and approximately 83% for DPPH. Furthermore, with increasing Prussian blue concentration, its catalase-like activity increases, promoting greater oxygen production. This indicates that the antioxidant mimicry activity of the Prussian blue nanozyme is concentration-dependent; higher concentrations result in stronger activity.

[0170] 4. Safety assessment of T-USPB-C at the cellular level

[0171] After staining with a live / dead staining solution and observation under a fluorescence microscope, the results are as follows: Figure 3 As can be seen, none of the three types of nanoparticles produced significant toxicity to cells, and almost no apoptotic cells were observed in the field of view, confirming that the nanoparticles do not damage cell viability.

[0172] For CCK-8 detection Figure 3 b- Figure 3 The results showed that after co-incubating cells with different concentrations of nanoparticles for 24 hours, 100 μg / mL and 200 μg / mL of T-USPB-C had a certain impact on cell viability.

[0173] Therefore, a T-USPB-C concentration of 50 μg / mL was selected for subsequent cell experiments. The concentrations of tirofiban and T-PB nanoparticles were calculated based on the tirofiban:Prussian blue:CREKA peptide ratio of 1:2:1 used in the nanoparticle synthesis process.

[0174] 5. Uptake of T-USPB-C by cardiomyocytes

[0175] After 24 hours, Cy5-labeled T-USPB-C was able to efficiently enter H9C2 cardiomyocytes, such as... Figure 3 d, through Figure 3 d. This fully demonstrates their therapeutic potential. Due to their highly efficient cellular uptake, the required drug dosage is reduced, thereby decreasing drug toxicity to other tissues and improving biocompatibility—factors crucial for clinical application. These nanozymes can penetrate cell membranes and enter intracellular sites prone to oxidative stress (such as mitochondria), making them significantly more effective at scavenging reactive oxygen species compared to nanozymes that remain only on the cell surface. Their tiny size enables them to penetrate cells; this ability, combined with their thrombus-targeting mechanism, allows them to accumulate at the thrombus site in cases such as myocardial infarction before entering surrounding cardiomyocytes, thus mitigating damage and achieving a synergistic therapeutic effect. Because they can remain intracellularly for extended periods, their antioxidant effects are sustained, effectively addressing continuous oxidative stress.

[0176] Overall, this highly efficient cellular uptake demonstrates their feasibility as potent intracellular antioxidants and provides evidence for their multiple functions in the treatment of complex heart diseases.

[0177] 6. Antioxidant effects of T-USPB-C at the cellular level

[0178] pass Figure 4 Results a and 4c showed that H9C2 cells and HUVECs stimulated by H / R accumulated more ROS compared to normal cells. Tirofiban demonstrated the ability to specifically clear ROS, consistent with previous findings on renal ischemia-reperfusion injury, namely that tirofiban reduces ROS generation, apoptosis, and leukocyte infiltration.

[0179] Compared to the T-USPB treatment group, the T-USPB-C treatment group showed a further reduction in ROS levels, highlighting the superior efficacy of T-USPB-C in ROS clearance. Figure 4 b and Figure 4d. By powerfully scavenging reactive oxygen species (ROS), it can directly break the vicious cycle of "ROS-endothelial damage-platelet activation" in the process of coronary microthrombus formation, reducing the thrombus burden from the source and exerting an antithrombotic effect. On the other hand, it can directly target the core of oxidative stress during myocardial ischemia-reperfusion, reduce myocardial cell damage, and thus significantly alleviate myocardial ischemia-reperfusion injury (MIRI), providing dual protection for cardiac function and fully demonstrating its significant value in the treatment of related heart diseases.

[0180] 7. Antithrombotic effect of T-USPB-C

[0181] This invention establishes a mouse carotid artery thrombosis model to evaluate the antiplatelet effect of the FeCl3-containing T-USPB-C complex; it also utilizes hematoxylin and eosin (HE) staining to assess the antithrombotic properties of the complex. Tirofiban, T-USPB, and T-USPB-C were administered to experimental animals via tail vein injection. Twenty-four hours after treatment, the mouse carotid arteries were removed and stained with HE to observe thrombus formation. In contrast, FeCl3 induced strong thrombus formation in the control group; while tirofiban and the two nanoparticle formulations inhibited thrombus formation to varying degrees, with T-USPB-C showing the most significant effect. At a dose of 90 μg / kg, the standard dose of tirofiban indeed reduced arterial thrombus formation. Figure 5 a and Figure 5 (b) As can be seen, T-USPB, by utilizing the ROS-clearing ability of Prussian blue, effectively disrupts the microthrombus formation mechanism induced by oxidative stress after coronary reperfusion, thus exhibiting more significant therapeutic effects. More importantly, T-USPB-C outperformed the other two groups. This advantage is directly attributed to the action of the CREKA peptide: this peptide can accumulate at the thrombus formation site, thereby enhancing local antiplatelet activity, ensuring maximum effect where it is most needed, while minimizing adverse reactions.

[0182] This targeting mechanism fully demonstrates the key advantages of T-USPB-C, specifically its ability to precisely deliver two dual-function components, tirofiban and Prussian blue, to the site of thrombus formation. This synergistic effect was further validated by DHE staining of frozen carotid artery tissue. FeCl3 treatment induced strong red fluorescence in the thrombus and vascular endothelial cells, indicating severe oxidative stress in these tissues. Although all three treatment methods reduced fluorescence intensity, the nanozyme activity of Prussian blue, similar to SOD, POD, and CAT, allowed for more efficient delivery. Figure 5 As can be seen, the T-USPB group and the T-USPB-C group were significantly more effective than tirofiban alone.

[0183] Of particular note is that, through Figure 5 As can be seen, T-USPB-C achieved the most significant ROS clearance effect because the CREKA-mediated targeting mechanism allows Prussian blue to accumulate in the thrombus microenvironment, thereby maximizing its effect in alleviating oxidative stress. These results fully demonstrate the great potential of T-USPB-C: its targeted delivery mechanism can enhance antiplatelet activity and ROS clearance at the thrombus site, thus more effectively breaking the vicious cycle of oxidative stress and thrombus expansion. This dual mechanism of action makes T-USPB-C a highly promising treatment, especially in cases such as myocardial infarction after PCI, where precision and versatility are crucial for reducing the risk of thrombus recurrence and protecting vascular function.

[0184] 8. T-USPB-C reduces MIRI and restores heart function

[0185] pass Figure 6 a and Figure 6 As can be seen, echocardiographic assessment at 28 days showed significant differences in functional recovery: cardiac function (EF) (16.08% ± 5.880%) and ejection fraction (FS) (6.965% ± 2.497%), hallmarks of severe left ventricular systolic dysfunction, significantly decreased in the I / R group, while cardiac function (EF) (57.96% ± 3.030%) increased almost threefold and ejection fraction (FS) (30.91% ± 2.569%) increased fourfold in the T-USPB-C group. This significant improvement cannot be attributed to tirofiban alone, as the tirofiban-only groups showed only minor functional improvements. The limitations of tirofiban may stem from its inability to address the broader pathological cascade of MIRI: its anti-inflammatory, anti-apoptotic, and reactive oxygen species scavenging abilities are weak, and its short half-life makes it unable to maintain therapeutic efficacy in the dynamic myocardial microenvironment after I / R. In contrast, T-USPB showed moderate improvement, highlighting the key role of Prussian blue in amplifying the efficacy of tirofiban by inhibiting oxidative stress and inflammation, two key drivers of persistent myocardial damage.

[0186] TTC staining further clarified the hierarchy of this treatment effect. The significant ischemic area (33.12% ± 3.746%) in the I / R group reflected extensive myocardial necrosis, while tirofiban moderately reduced this area by targeting thrombus-related perfusion problems (22.24% ± 1.488%), but did not improve downstream oxidative and inflammatory sequelae, such as... Figure 6 d and Figure 6 e.

[0187] T-USPB further extends this benefit (16.28% ± 1.112%) through the nanozyme activity of USPB, which disrupts ROS-mediated damage and enhances tissue survival after reperfusion. However, T-USPB-C achieves the smallest ischemic area (10.34% ± 0.8749%). This significant reduction highlights the synergistic value of targeted delivery: CREKA's affinity for thrombi and damaged tissue ensures concentrated co-localization of tirofiban (antithrombotic) and USPB (antioxidant / anti-inflammatory) at the ischemic core, maximizing their local effects while minimizing systemic exposure.

[0188] Overall, these data suggest that MIRI requires a spatially precise, multimodal intervention to break the cycle of thrombosis, oxidative stress, inflammation, and tissue necrosis. T-USPB-C, by combining the antiplatelet activity of tirofiban, the cytoprotective properties of Prussian blue, and the target specificity of CREKA, not only reduced ischemic injury but also achieved near-physiological levels of cardiac function recovery. This marks a paradigm shift: T-USPB-C does not treat the individual components of MIRI in isolation but rather as an integrated network to address the pathological process, making it a transformative candidate for clinical translation in post-ischemic heart disease.

[0189] In vivo experimental data collectively demonstrate that T-USPB-C is a multifunctional therapeutic agent. It exhibits excellent performance in anti-fibrosis, promoting angiogenesis, and anti-oxidation, advantages that distinguish it from tirofiban or T-USPB alone, and it is also more effective in alleviating myocardial ischemia-reperfusion injury. Figure 7 .

[0190] Regarding the anti-fibrotic effect, after 28 days of Masson's trichrome staining and Sirius Red staining, a clear difference in the intensity of action was observed: the I / R group showed extensive myocardial fibrosis, which is an inevitable result of cardiomyocyte death and fibroblast activation, while T-USPB-C significantly reduced the degree of fibrosis. Figure 7 a and Figure 7 b. This superior effect compared to T-USPB and tirofiban further highlights the key role of CREKA peptides. CREKA peptides can target thrombus-rich and damaged areas of the myocardium, thereby promoting the local accumulation of T-USPB-C at the lesion site, allowing its anti-fibrotic mechanism (likely related to USPB's inhibition of oxidative stress-induced fibroblast activation) to be fully exerted at the lesion site. This targeted mode of action more effectively disrupts the fibrotic process than non-targeted agents, thus preventing excessive extracellular matrix deposition and protecting the structure of the heart.

[0191] The antioxidant advantages of T-USPB-C were fully demonstrated in the DHE staining results three days after treatment. Its superoxide anion accumulation was significantly reduced, an effect even surpassing that of T-USPB and tirofiban. This is attributed to the synergistic effect between the nanozyme activity of USPB (which mimics the effects of SOD, POD, and CAT) and the targeting action of CREKA peptides. After accumulating at the lesion site, T-USPB-C can locally scavenge reactive oxygen species, thereby disrupting the oxidative stress cycle leading to cardiomyocyte death, inflammation, and fibrosis. This targeted antioxidant effect is crucial: it not only protects surviving cardiomyocytes from further damage but also creates a microenvironment conducive to angiogenesis and tissue regeneration.

[0192] Regarding the promotion of angiogenesis, the increased α-SMA fluorescence intensity in the infarct margin region of the T-USPB-C group directly reflects enhanced arteriolar formation, such as... Figure 7 e and Figure 7 f. This effect is likely the result of multiple factors working together: Prussian blue is believed to activate the HIF1-α pathway, which upregulates pro-angiogenic factors such as VEGF; combined with the targeting effect of CREKA peptide, this mechanism can significantly enhance vascular repair in the ischemic core region. Unlike tirofiban, which lacks pro-angiogenic effects, or T-USPB, which needs to circulate systemically to exert its effects, T-USPB-C can precisely deliver pro-angiogenic signals to the most critical sites of new blood vessel formation. This targeted angiogenesis can improve blood perfusion in the infarcted area and limit adverse pathological remodeling by providing structural support to the myocardium, thereby helping to maintain long-term cardiac function.

[0193] These findings fully demonstrate that T-USPB-C, through its targeted delivery mechanism, can coordinate multiple effects, including anti-fibrosis, angiogenesis promotion, and antioxidant activity. It intervenes in multiple pathological features of myocardial ischemia-reperfusion injury in a spatially precise manner, not only alleviating individual symptoms but also reversing the gradual deterioration of cardiac function, providing a comprehensive treatment strategy for ischemic heart disease.

[0194] 9. The inflammatory regulatory effect of T-USPB-C

[0195] The remarkable anti-inflammatory efficacy of T-USPB-C nanoparticles stems from the synergistic effects of targeted delivery, nanozyme regulation, and immune cell reprogramming. HE staining revealed extensive inflammatory cell infiltration and myocardial fiber rupture and necrosis in the I / R group's myocardial injury area, while T-USPB-C treatment significantly reduced inflammatory cell infiltration and maintained good myocardial fiber integrity. Western blotting confirmed that T-USPB-C reduced the release of pro-inflammatory factors such as IL-6 and TNF-α. Furthermore, T-USPB-C reduced cardiomyocyte apoptosis by upregulating Bcl-2 and downregulating Bax.

[0196] The remarkable anti-inflammatory effects of T-USPB-C nanoparticles stem from the synergistic effects of targeted delivery, nanozyme regulation, and immune cell reprogramming, which work together to combat multifaceted inflammation associated with myocardial ischemia-reperfusion injury (MIRI). In this invention, myocardial tissue from mice in different treatment groups was observed in detail using HE staining, such as... Figure 8 a. The results showed that in the ischemia-reperfusion (I / R) group, the myocardial injury area of ​​mice exhibited extensive inflammatory cell infiltration, as well as significant myocardial fiber rupture and necrosis.

[0197] In contrast, after T-USPB-C treatment, the number of inflammatory cells infiltrating the myocardial injury area was significantly reduced, and the integrity of the myocardial fibers was well preserved, such as... Figure 8 b. This demonstrates the significant effect of T-USPB-C in reducing myocardial inflammatory damage.

[0198] CREKA peptides promote the accumulation of T-USPB-C in the ischemic core region, which is rich in microthrombi and inflammatory cells, thus avoiding the systemic dilution that limits the efficacy of non-targeted therapies. By reducing microthrombi, CREKA improves perfusion and disrupts the pro-inflammatory cycle, and Western blotting confirms a reduction in the release of these cytokines, such as... Figure 8 c. According to HE staining results, this inhibition of microthrombi restored blood supply to myocardial tissue, reduced further ischemia-induced damage, and compensated for the reduction in inflammatory cell infiltration.

[0199] The USPB nanozyme-induced polarization of macrophages from a pro-inflammatory M1 phenotype to a reparative M2 phenotype is crucial. M1 macrophages produce reactive oxygen species (ROS), proteases, and cytokines, exacerbating damage, while M2 macrophages secrete anti-inflammatory factors and pro-angiogenic growth factors. The signs of myocardial tissue repair observed by HE staining are primarily attributed to this macrophage phenotypic shift; the repair function of M2 macrophages is morphologically manifested, such as the gradual restoration of myocardial fiber structure. Compared to the control group, the T-USPB-C group showed a more significant reduction in iNOS+ (M1 type) macrophages and a more pronounced repair effect, highlighting the targeted delivery mechanism. Furthermore, T-USPB-C reduces cardiomyocyte apoptosis by upregulating Bcl-2 and downregulating Bax. Figure 8 d, thereby limiting the release of damage-related molecular patterns and aseptic inflammation, further illustrating its role in protecting cardiomyocytes.

[0200] Overall, T-USPB-C can suppress early inflammation, prevent neutrophil infiltration, oxidative stress, and fibrosis, while synergistically counteracting the multiple impacts of MIRI with its pro-angiogenic and antioxidant effects. This holistic treatment approach is superior to monotherapy. Combined with the histomorphological improvements observed by HE staining, this fully demonstrates the transformative effect of T-USPB-C, which can block inflammation at multiple stages, thereby protecting myocardial structure, function, and long-term prognosis, and enhancing its clinical application potential.

[0201] 10. In vivo imaging assessment of the thrombus-targeting ability of T-USPB-C

[0202] pass Figure 9 As can be seen, compared with formulations lacking targeting function, the T-USPB-C group of this invention exhibits significantly stronger fluorescence signals in the thrombus region, demonstrating its enhanced thrombus-targeting ability. This targeting capability allows T-USPB-C to concentrate its action on the lesion site, reducing non-specific distribution in normal tissues, thereby improving therapeutic efficacy while minimizing potential side effects.

[0203] 11. Biocompatibility

[0204] pass Figure 10 It is evident that no significant signs of cumulative toxicity were observed in any organ with T-USPB-C, indicating that the prepared T-USPB-C is non-toxic or has extremely low toxicity.

[0205] 12. Conclusion

[0206] The T-USPB-C nanoparticles of this invention achieve targeted therapy via CREKA peptides and precise thrombus localization through specific fibrin recognition, avoiding systemic drug dispersion, reducing off-target effects, and increasing concentration at the lesion site to enhance efficacy. The ultra-small Prussian blue in T-USPB-C acts as an effective ROS scavenger, alleviating oxidative stress (a key driver of functional impairment) and protecting cardiomyocytes by neutralizing excess ROS. The loaded tirofiban synergistically inhibits platelet aggregation, clears microthrombi, alleviates MVO, and restores myocardial perfusion. T-USPB-C also promotes angiogenesis by improving the inflammatory microenvironment and stimulating pro-angiogenic factors to enhance blood supply to ischemic areas, while polarizing macrophages from pro-inflammatory M1 to anti-inflammatory M2 to reduce inflammation and promote tissue repair. This synergistic effect provides a new approach and method for sequential treatment of myocardial infarction with MVO and MIRI.

[0207] Example 5

[0208] Use of a type of tirofiban-loaded ultrasmall Prussian blue nanoparticle (T-USPB-C) in the preparation of drugs that reduce MVO and MIRI.

[0209] T-USPB-C targets the fibrin in thrombi. T-USPB-C delivers ultra-small Prussian blue nanoparticles carrying tirofiban to the thrombus site via the CREKA peptide, forming a high concentration of tirofiban locally. The Prussian blue in T-USPB-C scavenges pathological reactive oxygen species by mimicking enzyme activity, thereby disrupting the microthrombus formation mechanism induced by oxidative stress. The anti-inflammatory effect of Prussian blue in T-USPB-C reduces neutrophil infiltration.

[0210] Data from efficacy examples demonstrate that the T-USPB-C of this invention can carry tirofiban and exhibits nanozyme-like activities mimicking catalase and superoxide dismutase. Furthermore, its nanoscale size allows for efficient renal excretion, minimizing potential long-term toxicity. This T-USPB-C possesses excellent reactive oxygen species scavenging capabilities, rapidly improving microvascular perfusion through antithrombotic effects, and subsequently providing sustained protection through antioxidant and anti-inflammatory mechanisms, effectively preventing microvascular damage and microvascular injury-related MIRI.

[0211] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method of preparing tirofiban-loaded hyperfine superblue nanoparticles, characterized in that, Preparation of the ultra-small Prussian blue nanoparticles USPB by the following steps: S1, preparation of the ultra-small Prussian blue nanoparticles USPB by potassium ferricyanide, polyvinylpyrrolidone and hydrochloric acid-ethanol solution; S2, mixing and stirring tirofiban and the ultra-small Prussian blue nanoparticles USPB of S1 to obtain the tirofiban-loaded ultra-small Prussian blue T-USPB; S3, coupling the tirofiban-loaded ultra-small Prussian blue T-USPB of S2 with CREKA peptide by carbodiimide mediation to obtain the CREKA peptide-modified tirofiban-loaded Prussian blue T-USPB-C, which is the tirofiban-loaded ultra-small Prussian blue nanoparticles.

2. The method for preparing tirofiban-loaded ultrasmall Prussian blue nanoparticles according to claim 1, characterized in that, The S1 is specifically: dissolving potassium ferricyanide and polyvinylpyrrolidone in hydrochloric acid-ethanol solution reaction, then ultrafiltration purification of the concentrated solution, and washing and centrifuging the concentrated solution with ultrapure water to obtain the ultra-small Prussian blue nanoparticles USPB; The S2 is performed by the following steps: S2.1, dispersing the ultra-small Prussian blue nanoparticles USPB of S1 in ultrapure water A to obtain a mixed solution A; dissolving tirofiban in ultrapure water B to obtain a mixed solution B; S2.2, mixing and stirring the mixed solution A and the mixed solution B to react, then centrifuging, and washing the precipitate to obtain the tirofiban-loaded ultra-small Prussian blue T-USPB; The S3 is performed by the following steps: S3.1, dispersing the tirofiban-loaded ultra-small Prussian blue T-USPB obtained in S2 in MES buffer solution to enter S3.2; S3.2, adding a crosslinking agent composed of EDC and NHS to enter S3.3; S3.3, adding CREKA peptide, stirring to react, then centrifuging, and washing the precipitate to obtain the CREKA peptide-modified tirofiban-loaded Prussian blue T-USPB-C.

3. The method for preparing tirofiban-loaded ultrasmall Prussian blue nanoparticles according to claim 2, characterized in that: In the S1, the weight of potassium ferricyanide: the weight of polyvinylpyrrolidone: the volume of hydrochloric acid-ethanol solution is (15-40) mg: (500-1000) mg: (5-100) mL; The hydrochloric acid-ethanol solution is a hydrochloric acid-75% ethanol solution and the concentration of hydrochloric acid is 0.01M; In the S2, the weight of the ultra-small Prussian blue nanoparticles USPB: the volume of ultrapure water A: the weight of tirofiban: the volume of ultrapure water B is (15-25) mg: (10-100) mL: (5-15) mg: (10-100) mL; In the S3, the weight of the tirofiban-loaded ultra-small Prussian blue T-USPB: the volume of MES buffer solution: the weight of the crosslinking agent: the weight of CREKA peptide is (5-15) mg: (5-50) mL: (20-40) mg: (5-20) mg; In the crosslinking agent, the molar ratio of EDC to NHS is (0.1-10):

1.

4. The method for preparing tirofiban-loaded ultrasmall Prussian blue nanoparticles according to claim 3, characterized in that: In the S1, the weight of potassium ferricyanide: the weight of polyvinylpyrrolidone: the volume of hydrochloric acid-ethanol solution is 27.5 mg: 750 mg: 10 mL; In the S2, the weight of the ultra-small Prussian blue nanoparticles USPB: the volume of ultrapure water A: the weight of tirofiban: the volume of ultrapure water B is 20 mg: 20 mL: 10 mg: 20 mL; In the S3, the weight of the tirofiban-loaded ultra-small Prussian blue T-USPB: the volume of MES buffer: the weight of cross-linking agent: the weight of CREKA peptide is 10 mg: 10 mL: 32 mg: 10 mg; In the cross-linking agent, the molar ratio of EDC and NHS is 1:

1.

5. The method for preparing tirofiban-loaded ultrasmall Prussian blue nanoparticles according to claim 3, characterized in that, The S1 is specifically: dissolving potassium ferricyanide and polyvinylpyrrolidone in hydrochloric acid-ethanol solution for 4h-10h, then ultrafiltration under the condition of 10kDa molecular weight cut-off, then centrifugation under the centrifugal force of 3000g-8000g for 10min-60min to reserve the concentrated solution, and then centrifugation after washing the concentrated solution with ultrapure water to obtain the ultra-small Prussian blue nanoparticles USPB; The S2.2 is specifically: mixing the mixed solution A and the mixed solution B, stirring at room temperature for 10h-24h, then centrifugation, and washing the precipitate to obtain the tirofiban-loaded ultra-small Prussian blue T-USPB; The S3.2 is specifically: adding the cross-linking agent composed of EDC and NHS, activating at room temperature for 10min-60min, and then entering S3.3; The S3.3 is specifically: adding CREKA peptide, stirring for 10h-24h, centrifugation of the reactants under 8000g-12000g for 10min-60min, then washing the precipitate with ultrapure water to obtain the CREKA peptide-modified tirofiban-loaded Prussian blue T-USPB-C.

6. The method for preparing tirofiban-carrying epirubicin super- blue nanoparticles according to any one of claims 1 to 5, characterized in that: The nanoscale size of the ultra-small Prussian blue nanoparticles USPB is less than 10nm. The concentration of the MES buffer is 0.1M and the pH value is 6.

0.

7. A tirofiban-carrying epirubicin-hyperblue nanoparticle, characterized by: The tirofiban-loaded ultra-small Prussian blue nanoparticles are prepared by the preparation method of the tirofiban-loaded ultra-small Prussian blue nanoparticles according to any one of claims 1-6.

8. Use of tirofiban-loaded superparamagnetic blue nanoparticles for the preparation of a medicament for reducing MVO and MIRI, characterized by: The tirofiban-loaded ultra-small Prussian blue nanoparticles are prepared by the preparation method of the tirofiban-loaded ultra-small Prussian blue nanoparticles according to any one of claims 1-6.

9. Use of tirofiban-carrying epirubicin supraplatin nanoparticles according to claim 8 for the preparation of a medicament for reducing MVO and MIRI, characterized by: The tirofiban-loaded ultra-small Prussian blue nanoparticles target the fibrin of thrombus.

10. Use of tirofiban-carrying epirubicin supraplatin nanoparticles according to claim 8 for the preparation of a medicament for reducing MVO and MIRI, characterized by: In the tirofiban-loaded ultra-small Prussian blue nanoparticles, the tirofiban-loaded ultra-small Prussian blue nanoparticles are targeted and delivered to the thrombus site by the CREKA peptide, and a high concentration of tirofiban is formed locally; In the tirofiban-loaded ultra-small Prussian blue nanoparticles, the Prussian blue removes pathological active oxygen by simulating the activity of enzymes, thereby breaking the microthrombosis mechanism triggered by oxidative stress; In the tirofiban-loaded ultra-small Prussian blue nanoparticles, the anti-inflammatory effect of the Prussian blue reduces the infiltration of neutrophils.

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