Bionic nano system for treating acute ischemic stroke and application thereof

Abstract

The invention discloses a bionic nano system for treating acute ischemic stroke and application of the bionic nano system. According to the preparation method disclosed by the invention, the TNK-DNaseI (at) ZIF-8 nano-particles are prepared by loading the ZIF-8 with the TNK-DNaseI (at) ZIF-8 and then the TNK-DNaseI (at) ZIF-8 nano-particles are coated with the natural platelet membrane, so that the TNK-DNaseI (at) ZPM bionic nano-system is prepared. The bionic nano system can achieve efficient thrombolysis, obviously reduce the infarct area, improve neurological impairment and reduce bleeding complications. The invention provides a new drug choice for treating the acute ischemic stroke, and has a wide application prospect in the aspects of clinically treating the acute ischemic stroke and improving the prognosis of a patient with the acute ischemic stroke.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a bionic nanosystem for treating acute ischemic stroke and applications thereof. Background Art

[0002] Acute ischemic stroke (AIS) refers to a clinical syndrome characterized by impaired cerebral blood supply due to various cerebrovascular lesions, leading to localized tissue ischemia, hypoxia, and necrosis, and the rapid onset of corresponding neurological deficits. It is the second leading cause of death and the leading cause of disability in adults worldwide. China is one of the countries with the highest burden of AIS worldwide, and with the accelerating aging of its population, the incidence of AIS continues to increase. The key to acute treatment is to promptly re-open the occluded blood vessels, restore blood flow, and salvage the ischemic penumbra.

[0003] Recanalization methods for AIS include intravenous thrombolysis and endovascular mechanical thrombectomy. The latter is expensive, and although postoperative recanalization rates can exceed 90%, some patients still experience cerebral edema or hemorrhagic transformation after recanalization, often resulting in a poor prognosis. Intravenous thrombolysis is more widely used in clinical practice and remains the mainstay of treatment for AIS. Recombinant tissue plasminogen activator alteplase is an FDA-approved thrombolytic agent widely used for the treatment of AIS. However, its use is limited by a narrow therapeutic window, poor tissue specificity, a short half-life, and the risk of hemorrhagic complications. Tenecteplase (TNK), a genetically engineered third-generation thrombolytic agent based on alteplase, has higher fibrin specificity and a longer half-life. Numerous clinical studies have demonstrated that its reperfusion rate is higher than that of alteplase. Notably, the results of the EXTEND-IA-TNK study showed that although the reperfusion rate of TNK was twice that of alteplase, it was only 22%, indicating significant room for improvement.

[0004] Currently, thrombolytic drugs have disadvantages such as low bioavailability, off-target side effects, limited clot penetration, and increased risk of uncontrolled bleeding. Systemic administration and nonspecific activation of thrombolytic agents increase the likelihood of bleeding, limiting their use. In addition, protein-based thrombolytic drugs are rapidly inactivated upon injection into the bloodstream, necessitating increased drug dosages. Summary of the Invention

[0005] In order to overcome the above-mentioned defects and deficiencies in the prior art, the present invention provides a bionic nanosystem for treating acute ischemic stroke and its application.

[0006] The first object of the present invention is to provide a biomimetic nanosystem.

[0007] The second object of the present invention is to provide a method for preparing a bionic nanosystem.

[0008] The third object of the present invention is to provide the use of the above-mentioned bionic nanosystem in the preparation of drugs for treating acute ischemic stroke.

[0009] The fourth object of the present invention is to provide a drug for treating acute ischemic stroke.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0011] NETs (neutrophil extracellular traps) are unique extracellular network structures produced by neutrophils, primarily composed of DNA, histones, and granules. They have been shown to provide a platform for the binding of various coagulation components during AIS thrombosis. For example, NETs can form a scaffold that allows platelets and red blood cells to adhere to it in a concentrated, aggregated manner, participating in thrombosis. NETs' main components, DNA and histones, can synergize with the fibrin scaffold in the thrombus, making the thrombus more stable and more difficult to dissolve with drugs. Deoxyribonuclease I (DNase-I) is an endonuclease that can digest DNA and can degrade the DNA backbone of NETs. Previous studies by our team have shown that the combination of DNase I and tenecteplase can significantly enhance the therapeutic effect of acute ischemic stroke.

[0012] Furthermore, since DNase-I drugs have a short half-life in the circulation and unstable distribution, the safety and therapeutic effect of intravenous infusion are still unclear, and TNK has side effects such as bleeding complications, the present invention further studies and develops a nanocomposite material that simultaneously encapsulates tenecteplase and DNase-I to achieve precise, efficient and safe thrombus dissolution, which will have significant clinical significance for the treatment of AIS.

[0013] The present invention claims the following:

[0014] A biomimetic nanosystem, comprising an inner core and an outer membrane, wherein the inner core comprises tenecteplase, deoxyribonuclease I and an organic metal framework;

[0015] The outer membrane wraps around the outer periphery of the inner core.

[0016] Preferably, the organic metal framework is a zeolite imidazolate framework.

[0017] More preferably, the zeolitic imidazolate framework is ZIF-8.

[0018] Preferably, the outer membrane is a cell membrane.

[0019] More preferably, the cell membrane comprises a platelet membrane.

[0020] The preparation method of the bionic nanosystem comprises the following steps:

[0021] S1. Thoroughly mix tenecteplase, deoxyribonuclease I, 2-methylimidazole, zinc salt, surfactant, and ultrapure water to prepare TNK-DNase I@ZIF-8 nanoparticles;

[0022] S2. Add the outer membrane to the TNK-DNaseⅠ@ZIF-8 nanoparticle dispersion and mix thoroughly so that the outer membrane wraps the TNK-DNaseⅠ@ZIF-8 nanoparticles to prepare a biomimetic nanosystem.

[0023] Preferably, in step S1, the tenecteplase and deoxyribonuclease I are first mixed with 2-methylimidazole and ultrapure water, and then zinc salt is added and mixed, and finally a surfactant is added.

[0024] In order to prevent the metal ions contained in the solvent from causing DNase-I to convert to a positive charge and thus be unable to be encapsulated in the positively charged ZIF-8 material, the present invention uses ultrapure water as the solvent, and preferably, the ultrapure water is Milli-Q water.

[0025] Preferably, in step S1, the surfactant includes polyvinyl pyrrolidone.

[0026] Preferably, the zinc salt comprises zinc nitrate hexahydrate.

[0027] Preferably, in step S1, the mass ratio of tenecteplase to deoxyribonuclease I is 2.5-3.5:0.25-0.30.

[0028] More preferably, the mass ratio of tenecteplase to deoxyribonuclease I is 3:0.267.

[0029] More preferably, the molar ratio of the 2-methylimidazole, the zinc salt and the polyvinyl pyrrolidone is 2-3:4.5-5.5:0.02-0.03.

[0030] Further preferably, the molar ratio of the 2-methylimidazole, the zinc salt and the polyvinyl pyrrolidone is 2.5:5:3 / 111.1418.

[0031] Preferably, in step S2, the mass ratio of the TNK-DNaseⅠ@ZIF-8 nanoparticles to the outer membrane is 0.8-1.2:0.8-1.2.

[0032] More preferably, the mass ratio of the TNK-DNaseⅠ@ZIF-8 nanoparticles to the outer membrane is 1:1.

[0033] Preferably, in step S1, the time for thorough mixing is 15 to 25 minutes, and the temperature for thorough mixing is 24 to 26°C.

[0034] More preferably, in step S1, the time for sufficient mixing is 20 minutes.

[0035] Preferably, in step S2, the time for thorough mixing is 25 to 35 minutes, and the temperature for thorough mixing is 24 to 26°C.

[0036] More preferably, in step S2, the time for sufficient mixing is 30 minutes.

[0037] Preferably, after the outer membrane wraps the TNK-DNaseⅠ@ZIF-8 nanoparticles, the biomimetic nanosystem is obtained by an extrusion method.

[0038] Application of the above-mentioned bionic nanosystem in the preparation of drugs for treating acute ischemic stroke.

[0039] A drug for treating acute ischemic stroke, comprising the above-mentioned bionic nanosystem.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The present invention discloses a biomimetic nanosystem for treating acute ischemic stroke and its application. The biomimetic nanosystem can achieve efficient thrombolysis, significantly reduce infarct size, improve neurological deficits, and mitigate bleeding complications associated with tenecteplase, thereby reducing the risk of bleeding. This invention provides a new drug option for treating acute ischemic stroke and has broad application prospects in the clinical treatment of acute ischemic stroke and improving the prognosis of patients with acute ischemic stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1The particle size, Zeta potential, UV absorption spectrum and Fourier transform infrared spectrum of the nanoparticles; A: particle size of ZIF-8, TNK@ZIF-8 nanoparticles (T@Z), DNase-Ⅰ@ZIF-8 nanoparticles (D@Z), TNK-DNaseⅠ@ZIF-8 nanoparticles (TD@Z) and TNK-DNaseⅠ@ZPM biomimetic nanosystem (TD@ZPM); B: particle size of ZIF-8, TNK, DNase-Ⅰ, TNK@ZIF-8 nanoparticles (T@Z), DNase-Ⅰ@ZIF-8 nanoparticles (D@Z), TNK-DNaseⅠ@ZIF-8 nanoparticles (TD@Z) and TNK-DNaseⅠ@ZPM biomimetic nanosystem (TD@ZPM); Zeta potential of TNK-DNaseⅠ@ZIF-8 nanoparticles (T@Z), DNase-Ⅰ@ZIF-8 nanoparticles (D@Z), TNK-DNaseⅠ@ZIF-8 nanoparticles (TD@Z) and TNK-DNaseⅠ@ZPM biomimetic nanosystem (TD@ZPM), C: UV absorption spectra of TNK, ZIF-8 and TNK@ZIF-8 nanoparticles (T@Z), D: Fourier transform infrared spectroscopy results of TNK, ZIF-8 and TNK@ZIF-8 nanoparticles (T@Z).

[0043] Figure 2 Transmission electron microscopy images of ZIF-8, TNK@ZIF-8 nanoparticles (T@Z) and TNK-DNaseⅠ@ZPM biomimetic nanosystem (TD@ZPM).

[0044] Figure 3 Long-term stability of nanoparticles and expression of platelet membrane proteins; A: Long-term stability results of TNK-DNaseⅠ@ZIF-8 nanoparticles (TD@Z) and TNK-DNaseⅠ@ZPM biomimetic nanosystem (TD@ZPM), B: Expression of platelet membrane proteins CD41, CD61 and CD62p in mouse platelet membrane and TNK-DNaseⅠ@ZPM biomimetic nanosystem (TD@ZPM).

[0045] Figure 4 The release of TNK from TNK@ZIF-8 nanoparticles under different pH conditions.

[0046] Figure 5 Biocompatibility evaluation of TNK-DNaseⅠ@ZPM biomimetic nanosystem; A: Hemolysis test results, the upper left is the PBS group, the upper right is the TNK-DNaseⅠ@ZPM biomimetic nanosystem, and the lower left is the ddH2O group; B: Hemolysis rates of the PBS group, ddH2O group, and TNK-DNaseⅠ@ZPM biomimetic nanosystem (TD@ZPM) group; C: Platelet aggregation test results of the PBS group, PBS group containing thrombin, and PBS group containing TNK-DNaseⅠ@ZPM biomimetic nanosystem (TD@ZPM).

[0047] Figure 6These are the cytotoxicity test results of ZIF-8 and TNK-DNaseⅠ@ZIF-8 nanoparticles (TD@Z); A: ZIF-8, B: TNK-DNaseⅠ@ZIF-8 nanoparticles (TD@Z).

[0048] Figure 7 Statistical analysis of the in vitro thrombolysis test and thrombolysis rate results of patients in the PBS group, TNK group, TNK+DNase-Ⅰ group, TNK@ZIF-8 nanoparticles (T@Z) group with a pH of 6.4 or 7.4, TNK-DNaseⅠ@ZIF-8 nanoparticles (TD@Z) group with a pH of 6.4 or 7.4, and TNK-DNaseⅠ@ZPM biomimetic nanosystem (TD@ZPM) group with a pH of 6.4 or 7.4; A: representative results of the in vitro thrombolysis test, B: thrombolysis rate.

[0049] Figure 8 Figure 3 shows the statistical analysis of the in vitro thrombolysis test and thrombolysis rate of artificial blood clots in the PBS group, TNK group, TNK+DNase-Ⅰ group, TNK@ZIF-8 nanoparticles (T@Z) group at pH 6.4 or 7.4, TNK-DNaseⅠ@ZIF-8 nanoparticles (TD@Z) group at pH 6.4 or 7.4, and TNK-DNaseⅠ@ZPM biomimetic nanosystem (TD@ZPM) group at pH 6.4 or 7.4; A: representative results of the in vitro thrombolysis test, B: thrombolysis rate.

[0050] Figure 9 Representative graphs and statistical analysis of bleeding conditions after tail vein injection of different concentrations of TNK and TNK-DNaseⅠ@ZPM biomimetic nanosystem (TD@ZPM) in mice with photochemical thromboembolism model; A: Representative results of bleeding conditions, B: Statistical results of cerebral hemorrhage volume.

[0051] Figure 10 Figure 3. Laser speckle blood flow recovery and statistical analysis of FeCl3 carotid artery thrombosis model mice in normal saline group, TNK group, TNK+DNase-Ⅰ group, TNK@ZIF-8 nanoparticles (T@Z) group, TNK-DNaseⅠ@ZIF-8 nanoparticles (TD@Z) group, and TNK-DNaseⅠ@ZPM biomimetic nanosystem (TD@ZPM) group after administration; A: Laser speckle blood flow recovery, B: Relative cerebral blood flow.

[0052] Figure 11HE staining images of occluded blood vessels and statistical graphs of thrombus area in mice with FeCl3 carotid artery thrombosis model in normal group, normal saline group, TNK group, TNK+DNase-Ⅰ group, TNK@ZIF-8 nanoparticles (T@Z) group, TNK-DNaseⅠ@ZIF-8 nanoparticles (TD@Z) group, and TNK-DNaseⅠ@ZPM biomimetic nanosystem (TD@ZPM) group; A: HE staining image of occluded blood vessels, B: statistical graph of thrombus area.

[0053] Figure 12 Figure 3: Laser speckle blood flow recovery and statistical analysis of photochemical thromboembolism model mice in normal saline group, TNK group, TNK+DNase-Ⅰ group, TNK@ZIF-8 nanoparticles (T@Z) group, TNK-DNaseⅠ@ZIF-8 nanoparticles (TD@Z) group, and TNK-DNaseⅠ@ZPM biomimetic nanosystem (TD@ZPM) group after drug administration; A: Laser speckle blood flow recovery, relative cerebral blood flow.

[0054] Figure 13 Representative images of brain TTC staining, infarct volume, and statistical graphs of neurological deficit assessment at 24 h in mice with photochemical thromboembolism model in normal group, normal saline group, TNK group, TNK+DNase-Ⅰ group, TNK@ZIF-8 nanoparticles (T@Z) group, TNK-DNaseⅠ@ZIF-8 nanoparticles (TD@Z) group, and TNK-DNaseⅠ@ZPM biomimetic nanosystem (TD@ZPM) group; A: representative image of brain TTC staining, B: infarct volume, C: statistical graph of neurological deficit assessment.

[0055] Figure 14Figure 3 is a representative diagram of the thrombus targeting of nanoparticles in FeCl3 carotid artery thrombosis model mice and photochemical thromboembolism model mice, and a statistical diagram of the fluorescence signal changes over time; A: a representative diagram of the thrombus targeting of the FeCl3 carotid artery thrombosis model mice in the indocyanine green labeled TNK-DNaseⅠ@ZIF-8 nanoparticles (TD@Z) group and the TNK-DNaseⅠ@ZPM biomimetic nanosystem (TD@ZPM) group; B: a representative diagram of the thrombus targeting of the indocyanine green labeled TNK-DNaseⅠ@ZIF-8 nanoparticles (TD@Z) group and the TNK-DNaseⅠ@ZPM biomimetic nanosystem (TD@ZPM ) group in the photochemical thromboembolism model mice, C: Indocyanine green labeled TNK-DNaseⅠ@ZIF-8 nanoparticles (TD@Z) group and TNK-DNaseⅠ@ZPM biomimetic nanosystem (TD@ZPM) group in FeCl3 carotid artery thrombosis model mice fluorescence signal changes over time, D: Indocyanine green labeled TNK-DNaseⅠ@ZIF-8 nanoparticles (TD@Z) group and TNK-DNaseⅠ@ZPM biomimetic nanosystem (TD@ZPM) group in the photochemical thromboembolism model mice fluorescence signal changes over time.

[0056] Figure 15 Representative diagrams of organ distribution and fluorescence signal statistics of each organ of TNK-DNaseⅠ@ZPM biomimetic nanosystem (TD@ZPM) and TNK-DNaseⅠ@ZIF-8 nanoparticles (TD@Z) in two animal models 2 hours after administration; A: Representative diagram of organ distribution, PTS is the photochemical thromboembolism model, and FeCl3 is the FeCl3 carotid artery thrombosis model; B: Statistical diagram of fluorescence signals of each organ in mice with photochemical thromboembolism model; C: Statistical diagram of fluorescence signals of each organ in mice with FeCl3 carotid artery thrombosis model.

[0057] Figure 16 Representative pharmacokinetic graphs and normalized fluorescence intensity statistical graphs of TNK-DNaseⅠ@ZPM biomimetic nanosystem (TD@ZPM) and TNK-DNaseⅠ@ZIF-8 nanoparticles (TD@Z); A: representative pharmacokinetic graph, B: normalized fluorescence intensity statistical graph.

[0058] Figure 17 Representative images of HE staining of important organs and statistical graphs of key biochemical and coagulation indicators in blood of mice with photochemical thromboembolism model in the normal saline group, TNK group and TNK-DNaseⅠ@ZPM biomimetic nanosystem (TD@ZPM) group 7 days after drug administration; A: Representative images of HE staining of important organs, B: Statistical graph of key biochemical and coagulation indicators in blood.

[0059] Figure 18is the Zeta potential of DNase I in different solvents. Solution 1 is pure water, and Solution 2 is ultrapure water. DETAILED DESCRIPTION

[0060] The present invention is further described below with reference to specific examples, which, however, are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0061] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0062] Example 1 Preparation and Characterization of Nanoparticles

[0063] 1. Experimental Methods

[0064] 1. Preparation of Nanoparticles

[0065] Preparation of TNK@ZIF-8 nanoparticles (T@Z): 3 mg of TNK was mixed with 1 mL of 2-methylimidazole (2.5 mmol) aqueous solution (prepared with Milli-Q ultrapure water), and then 0.1 mL of Zn(NO3)2·6H2O (5 mmol) was added. The mixture was mixed with 3 mg of PVP and stirred at room temperature for 20 min. The mixture was centrifuged at 10,000 rpm for 10 min, washed three times with Milli-Q ultrapure water to remove the residue, and vacuum dried to obtain TNK@ZIF-8 nanoparticles.

[0066] Preparation of DNase-Ⅰ@ZIF-8 nanoparticles (D@Z): 300 μg of DNase-Ⅰ was mixed with 1 mL of 2-methylimidazole (2.5 mmol) aqueous solution (prepared with Milli-Q ultrapure water), and then 0.1 mL of Zn(NO3)2·6H2O (5 mmol) was added. The mixture was mixed with 3 mg of PVP and stirred at room temperature for 20 min. The mixture was centrifuged at 10,000 rpm for 10 min, washed three times with Milli-Q ultrapure water to remove the residue, and vacuum dried to obtain DNase-Ⅰ@ZIF-8 nanoparticles.

[0067] Preparation of TNK-DNaseⅠ@ZIF-8 nanoparticles (TD@Z): 3 mg of TNK, 267 μg of DNase-Ⅰ and 1 mL of 2-methylimidazole (2.5 mmol) aqueous solution (prepared with Milli-Q ultrapure water) were mixed, and then 0.1 mL of Zn(NO3)2·6H2O (5 mmol) was added. The mixture was mixed with 3 mg of PVP and stirred at room temperature for 20 min. The mixture was centrifuged at 10,000 rpm for 10 min, washed three times with Milli-Q ultrapure water to remove the residue, and vacuum dried to obtain TNK-DNaseⅠ@ZIF-8 nanoparticles.

[0068] Preparation of the TNK-DNase I@ZPM biomimetic nanosystem (TD@ZPM): Platelet membranes were extracted from C57BL / 6J mice and prepared by freeze-thawing. A 1 mg / mL platelet membrane solution was added to an equal volume of a 1 mg / mL TNK-DNase I@ZIF-8 biomimetic nanosystem dispersion (prepared in Milli-Q ultrapure water) and thoroughly mixed for 30 minutes. The membranes were then extruded into 800, 400, and 200 nm polycarbonate porous membranes using an Avanti microliposome extruder. The membranes were centrifuged at 10,000 g for 5 minutes, and the pellets were resuspended in water to obtain the TNK-DNase I@ZPM biomimetic nanosystem.

[0069] 2. Characterization of Nanoparticles

[0070] According to the total mass, dosage and amount of protein encapsulated in the TNK@ZIF-8 nanoparticles and DNase-Ⅰ@ZIF-8 nanoparticles prepared in step 1, the encapsulation efficiency of ZIF-8 for TNK and DNase-Ⅰ and the drug loading efficiency of the nanoparticles were calculated.

[0071] The Zetasizer Nano ZS particle analyzer was used to characterize the particle size and Zeta potential of ZIF-8, TNK, DNase-Ⅰ, as well as the TNK@ZIF-8 nanoparticles, DNase-Ⅰ@ZIF-8 nanoparticles, TNK-DNaseⅠ@ZIF-8 nanoparticles and TNK-DNaseⅠ@ZPM biomimetic nanosystems prepared in step 1.

[0072] The UV absorption spectra and Fourier transform infrared spectra of ZIF-8, TNK and TNK@ZIF-8 nanoparticles prepared in step 1 were observed using a UV-visible spectrophotometer and a Fourier transform infrared spectrometer.

[0073] Transmission electron microscopy was used to observe the morphology of ZIF-8, TNK@ZIF-8 nanoparticles prepared in step 1, and TNK-DNaseⅠ@ZPM biomimetic nanosystem.

[0074] The TNK-DNaseⅠ@ZIF-8 nanoparticles and TNK-DNaseⅠ@ZPM biomimetic nanosystems prepared in step 1 were stored in PBS buffer at room temperature for 7 days. The size of the nanoparticles was measured using dynamic light scattering every day to evaluate the long-term stability of the nanoparticles.

[0075] Western blot was used to detect the expression of key platelet membrane proteins (CD41, CD61 and CD62p) in the platelet membrane and the TNK-DNaseⅠ@ZPM biomimetic nanosystem prepared in step 1.

[0076] The TNK@ZIF-8 nanoparticles prepared in step 1 were dispersed in PBS buffer solutions with pH values ​​of 6.4 and 7.4, respectively, and the amount of TNK released under different pH conditions was detected.

[0077] The biocompatibility of the TNK-DNaseⅠ@ZIF-8 nanoparticles prepared in step 1 was evaluated using hemolysis assay and platelet aggregation assay; the cytotoxicity of the TNK-DNaseⅠ@ZIF-8 nanoparticles prepared in step 1 was evaluated using the CCK-8 method.

[0078] 2. Experimental Results

[0079] 1. Encapsulation efficiency and drug loading

[0080] According to the results in Table 1, the encapsulation efficiency of ZIF-8 for TNK is 73.19%, and the drug loading efficiency is 10.4%. The encapsulation efficiency of ZIF-8 for DNase-Ⅰ is 82.40%, and the drug loading efficiency is 1.65%.

[0081] Table 1 Encapsulation efficiency and drug loading efficiency of ZIF-8 for TNK and DNase-Ⅰ

[0082] TNK@ZIF-8 DNase-Ⅰ@ZIF-8 Total mass (mg) 21.1 13.3 Dosage (mg) 3 0.267 Protein content (mg) 2.19565 0.22 Encapsulation efficiency (%) 73.19 82.40 Drug loading rate (%) 10.4 1.65

[0083] 2. Particle size and Zeta potential

[0084] The particle sizes of ZIF-8, TNK@ZIF-8 nanoparticles, DNase-Ⅰ@ZIF-8 nanoparticles, TNK-DNaseⅠ@ZIF-8 nanoparticles and TNK-DNaseⅠ@ZPM biomimetic nanosystems prepared in step 1 are as follows: Figure 1 As shown in Figure A, the particle size of ZIF-8 is 114.05±7.8nm, the particle size of TNK@ZIF-8 nanoparticles is 104.12±1.81nm, the particle size of DNase-Ⅰ@ZIF-8 nanoparticles is 181.08±17.97nm, and the particle size of TNK-DNaseⅠ@ZIF-8 nanoparticles is 155.35±28.94nm. The size is further increased after platelet membrane coating, and the final particle size of the TNK-DNaseⅠ@ZPM biomimetic nanosystem is 196.2±32.74nm.

[0085] The Zeta potential results of ZIF-8, TNK, DNase-Ⅰ and TNK@ZIF-8 nanoparticles, DNase-Ⅰ@ZIF-8 nanoparticles, TNK-DNaseⅠ@ZIF-8 nanoparticles and TNK-DNaseⅠ@ZPM biomimetic nanosystems prepared in step 1 are shown in Figure 2. Figure 1As shown in Figure B, the zeta potential of ZIF-8 is +12.53±0.91mV, that of TNK is -12.4±0.76mV, and that of DNase-Ⅰ is -3.34±0.80mV. Compared to unloaded ZIF-8, loading the negatively charged TNK payload reduces its surface zeta potential. After ZIF-8 encapsulates the two protein drugs, the zeta potential of TNK-DNaseⅠ@ZIF-8 nanoparticles drops to -14.7±0.82mV. The membrane coating also significantly shields the inherently positively charged ZIF-8 core, resulting in a greater negative charge of the TNK-DNaseⅠ@ZPM biomimetic nanosystem, at -19.63±1.16mV.

[0086] The results of particle size and Ztea potential preliminarily indicated that ZIF-8 could simultaneously load TNK and DNase-Ⅰ, and the platelet membrane had been successfully coated around the TNK-DNaseⅠ@ZPM biomimetic nanosystem.

[0087] 3. Ultraviolet absorption spectroscopy and Fourier transform infrared spectroscopy

[0088] The UV absorption spectrum results are as follows Figure 1 As shown in Figure C, the results show that due to the presence of TNK protein residues, TNK has an obvious absorption peak at about 278 nm. It is also observed that compared with ZIF-8, TNK@ZIF-8 nanoparticles have an absorption peak at 280-290 nm, which indicates that ZIF-8 has successfully loaded TNK protein.

[0089] The Fourier transform infrared spectroscopy results are as follows Figure 1 As shown in Figure D, the spectrum of TNK@ZIF-8 nanoparticles shows a -1 and 1550cm -1 The absorption peaks of 3470 cm-1 correspond to the TNK amide A band (NH stretching vibration) and amide II band (CN stretching vibration and NH bending vibration) appearing in TNK@ZIF-8 nanoparticles. -1 The peaks observed at the single bond correspond to the stretching vibration of the aromatic C-H bond. TNK@ZIF-8 nanoparticles also show the functional groups of ZIF-8. The stretching vibration of the imidazole ring is between 1350 and 1500 cm -1 Identification within the double bond range, at 600 cm -1 and 1500cm -1 Adsorption bands were observed between the two groups, indicating the bending vibration mode of the imidazole ring, which again demonstrated that TNK was successfully encapsulated in ZIF-8.

[0090] 4. Basic appearance

[0091] Transmission electron microscopy images Figure 2 The results show that the average diameter of ZIF-8 is 80 nm, while the ZIF-8 loaded with TNK protein exhibits a rhombic dodecahedral structure. Compared to ZIF-8, the hydrodynamic size of TNK@ZIF-8 nanoparticles increases slightly, reaching an average diameter of 110 nm. The TNK-DNaseⅠ@ZPM biomimetic nanosystem is elliptical overall and has a clear membrane structure, demonstrating that the platelet membrane is successfully coated with the TNK-DNaseⅠ@ZIF-8 nanoparticles.

[0092] 5. Long-term stability and platelet membrane protein retention

[0093] Long-term stability results such as Figure 3 As shown in Figure A, the results show that the particle size of the TNK-DNaseⅠ@ZPM biomimetic nanosystem remained essentially stable over 7 days. The particle size of the TNK-DNaseⅠ@ZIF-8 nanoparticles without platelet membrane coating increased slowly at first and then rapidly over time, with a significant increase observed after day 3. This result highlights the benefit of using a natural platelet membrane coating to stabilize nanoparticles under physiological conditions.

[0094] The expression of key platelet membrane proteins (CD41, CD61 and CD62p) in the platelet membrane and the TNK-DNaseⅠ@ZPM biomimetic nanosystem prepared in step 1 is shown in Figure 2. Figure 3 As shown in Figure B, the results show that the TNK-DNaseⅠ@ZPM biomimetic nanosystem retains the same protein bands as the platelet membrane, including CD41, CD61 and CD62p proteins, indicating that the membrane coating is successful, providing a prerequisite guarantee for the thrombus targeting ability of the TNK-DNaseⅠ@ZPM biomimetic nanosystem.

[0095] 6. Release of nanoparticles under different pH conditions

[0096] The results are as follows Figure 4 As shown in the data, under the condition of neutral pH 7.4, which is close to physiological conditions, the release rate of TNK@ZIF-8 nanoparticles is relatively slow, with the release amount within 0.17h (about 10min) being only 3.26%, the release amount within 1h being 12.31%, and the release amount within 4h being 18.33%. Even at 24h, the release amount of TNK is only 23.45%.

[0097] In contrast, TNK@ZIF-8 nanoparticles exhibited significant rapid release properties in a slightly acidic environment of pH 6.4, with the release amount rapidly increasing from the initial 0.29% to 6.24% in just 0.17 h. The TNK release amount reached 28.50% in 1 h, 58.18% in 4 h, and finally nearly complete release in 24 h, with the release amount reaching 98.88%.

[0098] The above results indicate that the pH-responsive release characteristics of TNK@ZIF-8 nanoparticles can protect TNK within the nanocarrier structure. After reaching the thrombus site, TNK can be effectively released through the acidic microenvironment.

[0099] 7. Biocompatibility

[0100] Hemolysis test results Figure 5 As shown in A and B, PBS buffer was used as negative control and ddH2O as positive control. The results showed that the TNK-DNaseⅠ@ZPM biomimetic nanosystem group ( Figure 5 A in the upper right) and PBS group ( Figure 5 The supernatant after centrifugation for 3 hours was clear and transparent, the morphology of red blood cells was normal, and the hemolysis rate was less than 5% ( Figure 5 B), indicating that the TNK-DNaseⅠ@ZPM biomimetic nanosystem does not produce hemolysis in the circulation.

[0101] Platelet aggregation test results Figure 5 As shown in Figure C, PBS buffer containing thrombin was used as a positive control, and PBS buffer was used as a negative control. The results showed that the TNK-DNaseⅠ@ZPM biomimetic nanosystem itself did not exhibit any platelet activation ability. The platelet aggregation rate in this group was similar to that in the PBS group, indicating that the TNK-DNaseⅠ@ZPM biomimetic nanosystem does not pose a risk of promoting thrombosis in the circulation.

[0102] The above results all indicate that the TNK-DNaseⅠ@ZPM biomimetic nanosystem has excellent biocompatibility in vivo.

[0103] 8. Cytotoxicity

[0104] CCK-8 results Figure 6 As shown in Figures A to B, ZIF-8 exhibited negligible cytotoxicity at concentrations below 100 μg / mL. When the concentration reached 200 μg / mL, the cell survival rate was only 50% ( Figure 6 A), this is because ZIF-8 itself is positively charged and toxic to cells.

[0105] TNK-DNaseⅠ@ZIF-8 nanoparticles did not show obvious cytotoxicity in a series of concentration gradients. At a concentration of 200 μg / mL, a cell survival rate of 120% was observed, showing a cell proliferation phenomenon ( Figure 6 B) in.

[0106] The above results show that TNK-DNaseⅠ@ZIF-8 nanoparticles have good safety.

[0107] Example 2 Effect of Nanoparticles on Thrombolysis in Vitro

[0108] 1. Experimental Methods

[0109] Fresh thrombi were collected from patients with AIS who underwent endovascular thrombectomy at the First Affiliated Hospital of Jinan University (Guangzhou Overseas Chinese Hospital) for in vitro thrombolysis experiments.

[0110] The artificial hematopoietic clots were obtained from healthy volunteers. A questionnaire confirmed no history of stroke, hematologic disorders, or other major medical conditions that could affect coagulation. The volunteers also avoided negative lifestyle habits such as smoking and alcohol abuse, and had not taken any medications that could affect coagulation in the past three months. Blood was collected using a 3.2 wt% sodium citrate anticoagulant tube. 16.6 mM anhydrous calcium chloride was added to 250 μL of blood and calcified at 37°C for 30 minutes to form a clot core. The remaining blood was centrifuged at 120 g for 15 minutes to obtain leukocyte-rich platelet-rich plasma (WRP). The clot core was then added to the WRP, and the mixture was mixed in a thermomixer at 37°C, 500 rpm, for 3.5 hours to form a complete clot.

[0111] Fresh thrombi or artificial blood clots from patients were transferred to 6-well plates pre-loaded with PBS, 1 μg / mL TNK solution, 1 μg / mL TNK + 100 IE / mL DNase-I solution, TNK@ZIF-8 nanoparticles (prepared in Example 1) containing 1 μg / mL TNK at pH 6.4 or 7.4, TNK-DNase I@ZIF-8 nanoparticles (prepared according to Example 1) containing 1 μg / mL TNK at pH 6.4 or 7.4, or TNK-DNase I@ZPM biomimetic nanosystem (prepared according to Example 1) containing 1 μg / mL TNK at pH 6.4 or 7.4. 1 μM plasminogen was added to each treatment group to ensure TNK activity. The plates were incubated at 37°C for 1 hour. The weight of each thrombus was measured at 0, 20, 40, and 60 minutes using an ultraprecision balance. The thrombus lysis rate was expressed as the percentage of the thrombus weight at each time point relative to the initial weight.

[0112] 2. Experimental Results

[0113] 1. In vitro thrombolysis of patients' thrombi

[0114] The results of the in vitro thrombolysis experiment of the patient's thrombus are as follows Figure 7 As shown in Figures A-B, the thrombus weight in the PBS group remained essentially unchanged over 60 minutes. Thrombi in both the TNK and TNK+DNase-Ⅰ groups were dissolved to varying degrees at 60 minutes, with the latter demonstrating greater dissolution efficiency, with the remaining thrombus weight being less than the initial weight (TNK+DNase-Ⅰ vs. TNK: 31.17% vs. 46.63%, p < 0.001). Thrombi dissolution varied across the TNK@ZIF-8 nanoparticle, TNK-DNaseⅠ@ZIF-8 nanoparticle, and TNK-DNaseⅠ@ZPM biomimetic nanosystem groups at varying pH values ​​and in the presence or absence of platelet coating.

[0115] At neutral pH 7.4, at 60 min, the thrombus weight in the TNK@ZIF-8 nanoparticle group remained almost unchanged, at approximately 92.62% of the initial weight, due to the low amount of TNK released. The thrombus weight in the TNK-DNaseⅠ@ZIF-8 nanoparticle group was approximately 87.25% of the initial weight. The thrombus weight in the TNK-DNaseⅠ@ZPM biomimetic nanosystem group decreased significantly compared with the TNK@ZIF-8 nanoparticle and TNK-DNaseⅠ@ZIF-8 nanoparticle groups, at approximately 68.73% of the initial weight (TNK-DNaseⅠ@ZIF-8 vs TNK@ZIF-8, p < 0.0001; TNK-DNaseⅠ@ZPM vs TNK-DNaseⅠ@ZIF-8, p < 0.0001).

[0116] Under slightly acidic conditions of pH 6.4, thrombus dissolution was more pronounced in each nanoparticle group. At 60 minutes, the thrombus weight in the TNK@ZIF-8 nanoparticle group was approximately 76.17% of the initial weight, while that in the TNK-DNaseⅠ@ZIF-8 nanoparticle group was reduced to 70.08% of the initial weight. Notably, the thrombus weight in the TNK-DNaseⅠ@ZPM biomimetic nanosystem group was only 27.28% of the initial weight, but this was not statistically different from the TNK+DNase-Ⅰ group (at 60 minutes, TNK-DNaseⅠ@ZPM vs. TNK+DNase-Ⅰ, p=0.13; TNK-DNaseⅠ@ZPM vs. the other groups, p<0.0001).

[0117] The above results show that compared with a neutral environment, the thrombus weight of each nanoparticle group decreased more under slightly acidic conditions, which corresponds to the property of ZIF-8 that it can only decompose and release drugs under acidic conditions. In addition, among all the groups, the TNK+DNase-Ⅰ group and the TNK-DNaseⅠ@ZPM biomimetic nanosystem (pH 6.4) group showed the most significant thrombolytic efficiency. Initially, the thrombus dissolution effect of the TNK-DNaseⅠ@ZPM biomimetic nanosystem lagged significantly behind that of the TNK+DNase-Ⅰ, possibly due to the slower and smaller release rate of the drug from ZIF-8 at the beginning. However, at 60 minutes, there was no statistical difference in the thrombolytic rate between the two groups. Moreover, according to the results of the TNK@ZIF-8 nanoparticles release of TNK under acidic conditions in Example 1, although the drug release rate of the TNK@ZIF-8 nanoparticles was only about 30% at 60 minutes, the thrombolytic effect was significant, which to some extent demonstrates the high efficiency of the TNK-DNaseⅠ@ZPM biomimetic nanosystem in thrombolysis.

[0118] 2. In vitro thrombolysis of artificial blood clots

[0119] Because the collected thrombi were from different patients and had heterogeneous components, it was impossible to objectively verify the thrombolytic efficacy of each group of drugs. Therefore, artificial blood clots were prepared using peripheral blood donated by healthy volunteers and treated in the same way as the patient thrombi. The results showed that the thrombolytic effect of the artificial blood clots was similar to that of the patients ( Figure 8 A-B): No clot dissolution was observed in the PBS group. At 60 minutes, clot weight in the TNK group was 51.38% of its initial weight, while that in the TNK + DNase-I group was 31.10% (TNK + DNase-I vs. TNK, p < 0.01), demonstrating that the addition of DNase-I significantly improved thrombolytic efficiency.

[0120] At pH 7.4, the artificial blood clots in the TNK@ZIF-8 nanoparticle group were almost not dissolved, with 92.43% of the artificial blood clot remaining. The artificial blood clot weight in the TNK-DNaseⅠ@ZIF-8 nanoparticle group was 85.50% of the initial weight, and the artificial blood clot weight in the TNK-DNaseⅠ@ZPM biomimetic nanosystem group was 66.3% of the initial weight (TNK-DNaseⅠ@ZIF-8 vs TNK@ZIF-8, p < 0.01; TNK-DNaseⅠ@ZPM vs TNK-DNaseⅠ@ZIF-8, p < 0.001).

[0121] In a slightly acidic environment (pH 6.4), the thrombolytic efficiency of each group of nanoparticles was improved. In the TNK@ZIF-8 nanoparticle group, 77.93% of the artificial blood clot remained, and in the TNK-DNaseⅠ@ZIF-8 nanoparticle group, the weight of the artificial blood clot was 70.52% of the initial weight. The TNK-DNaseⅠ@ZPM biomimetic nanosystem group had the highest thrombolytic efficiency, with the remaining artificial blood clot weight being only 28.22% of the initial weight (TNK-DNaseⅠ@ZIF-8 vs TNK@ZIF-8, p < 0.05; TNK-DNaseⅠ@ZPM vs TNK-DNaseⅠ@ZIF-8, p < 0.0001).

[0122] One difference from the results of the patient thrombus thrombolysis experiment is that the thrombolytic efficiency of the TNK-DNaseⅠ@ZPM bionic nanosystem (pH 6.4) group at 60 minutes was significantly higher than that of the TNK+DNase-Ⅰ group (TNK-DNaseⅠ@ZPM vs TNK+DNase-Ⅰ, p < 0.05). This result once again adds strong evidence for the efficient thrombolysis of the TNK-DNaseⅠ@ZPM bionic nanosystem.

[0123] Example 3 Effect of Nanoparticles on Thrombolysis in Vivo

[0124] 1. Experimental Methods

[0125] 1. Establishment of a Photochemical Thromboembolism Mouse Model

[0126] To determine the drug concentration for in vivo thrombolytic experiments and assess the bleeding risk of the TNK-DNaseⅠ@ZPM biomimetic nanosystem, a mouse model of photochemical thromboembolism was constructed. The following procedures were performed: Mice were anesthetized with 1% sodium pentobarbital solution (w / v) intraperitoneally. After shaving the head and exposing the left temporal muscle, a 2 mm diameter bone window was created in the region of the left middle cerebral artery. Rose Bengal solution (50 mg / kg) was injected via the tail vein. Five minutes later, a green laser beam (532 nm, 5 mW) was used for 16 minutes and 30 seconds. Two hours after vascular occlusion, TNK solutions were injected into the tail vein at concentrations of 2.5 mg / kg, 5.0 mg / kg, 7.5 mg / kg, and 10 mg / kg, respectively. Twenty-four hours later, the brains were perfused with saline and the intracerebral hemorrhage volume was calculated.

[0127] To determine the in vivo thrombolytic effect of nanoparticles, a photochemical thromboembolism mouse model was established according to the above method. Two hours after vascular occlusion, normal saline, TNK (2.5 mg / kg), TNK (2.5 mg / kg) + DNase-I (50 μg), TNK@ZIF-8 nanoparticles containing 2.5 mg / kg TNK (prepared according to Example 1), TNK-DNaseI@ZIF-8 nanoparticles containing 2.5 mg / kg TNK (prepared according to Example 1), or TNK-DNaseI@ZPM biomimetic nanosystem containing 2.5 mg / kg TNK (prepared according to Example 1) were injected into the tail vein. Laser speckle blood flow imaging was used to observe the recovery of cerebral blood flow in mice at baseline (preoperatively), immediately after surgery, and 0, 20, 40, and 60 minutes after administration. The relative cerebral blood flow (CBF) at a certain time point was expressed as a percentage of the CBF measured in that area at baseline.

[0128] Twenty-four hours after modeling, cerebral infarct volume was assessed in each group of mice using 2,3,5-triphenyltetrazole chloride (TTC) staining. Brains were removed directly from mice after anesthesia and quickly frozen at -20°C for approximately 20 minutes. Slices were then cut from the frontal to occipital poles at 2 mm intervals using a razor blade. The slices were placed in a well plate containing 2% TTC solution (w / v), covered with tin foil to protect from light, and then placed in a 37°C incubator for staining for 20–30 minutes, turning the slices once or twice to ensure uniform staining. The TTC solution was recovered, and the slices were fixed with 4% paraformaldehyde solution (w / v). The slices were removed, dried with filter paper, and arranged in order. The slices were then placed on a background plate for photography. Image-J software was used to calculate the proportion of the infarcted area to the total brain tissue.

[0129] The Longa five-point scoring system was used to assess neurological deficits in mice. Score 0: normal, no neurological deficit; score 1: inability to fully extend the affected forepaw; score 2: the mouse circles toward the paralyzed side when walking; score 3: the mouse falls toward the paralyzed side when walking; and score 4: inability to walk spontaneously and loss of consciousness.

[0130] 2. Establishment of FeCl3 Carotid Artery Thrombosis Mouse Model

[0131] Mice were anesthetized with an intraperitoneal injection of 1% sodium pentobarbital solution (w / v) and placed in the supine position. A 1-cm mid-cervical incision was made to expose the left common carotid artery and blunt dissection. The artery was covered with filter paper soaked in 10% FeCl₃ solution (w / v) for 10 minutes. After removal of the filter paper, the vessel was washed with sterile PBS to prevent residual FeCl₃ solution. The tail vein was then injected with normal saline, TNK (2.5 mg / kg), TNK (2.5 mg / kg) plus DNase-I (50 μg), or TNK@ZIF-8 nanoparticles containing 2.5 mg / kg TNK, TNK-DNase I@ZIF-8 nanoparticles containing 2.5 mg / kg TNK, or TNK-DNase I@ZPM biomimetic nanosystem containing 2.5 mg / kg TNK. Twenty minutes after administration, vascular recanalization was observed using laser speckle blood flow imaging. The occluded common carotid artery was harvested for hematoxylin and eosin staining, and the percentage of thrombus area relative to the entire vessel was calculated to assess the thrombolytic efficacy of each treatment group.

[0132] 2. Experimental Results

[0133] In the photochemical thromboembolism model animal, TNK concentration gradients of 2.5mg / kg, 5.0mg / kg, 7.5mg / kg and 10.0mg / kg were set up to compare the brain hemorrhage volume of mice under different concentrations. The results showed that the mortality rate of mice in the 10.0mg / kg TNK concentration group reached 80% 24 hours after administration, indicating that this concentration of TNK has poor in vivo safety and is not suitable as a dosage concentration for animal experiments. As the TNK concentration decreased from 7.5mg / kg to 2.5mg / kg, Figure 9 As shown in A to B in Figure 3, the hemorrhage volume in the mouse brain tissue gradually decreased (5.0 mg / kg vs 7.5 mg / kg: 10.39% vs 14.84%, p < 0.0001; 2.5 mg / kg vs 5.0 mg / kg: 8.63% vs 10.39%, p < 0.05). Considering that the thrombolytic effect of 2.5 mg / kg TNK combined with DNase-Ⅰ in previous studies was very ideal, this concentration was selected for the in vivo thrombolytic experiment.

[0134] The TNK-DNaseⅠ@ZPM biomimetic nanosystem containing 2.5 mg / kg TNK was given to mice with a photochemical thromboembolism model. The results showed that compared with the same concentration of TNK, the TNK-DNaseⅠ@ZPM biomimetic nanosystem containing 2.5 mg / kg TNK caused less cerebral hemorrhage in mice (TNK-DNaseⅠ@ZPM vs 2.5 mg / kg TNK: 5.58% vs 8.63%, p < 0.01).

[0135] In the FeCl3 carotid artery thrombosis model mice, laser speckle blood flow imaging showed that the carotid artery blood flow of each group of mice recovered to varying degrees after drug administration. Among them, the TNK-DNaseⅠ@ZPM biomimetic nanosystem group mice showed the most significant recovery of occluded blood flow in a short period of time compared with the other groups ( Figure 10 A~B in the figure) can recover to 77.80% of the initial blood flow on average, while the carotid artery blood flow of TNK+DNase-Ⅰ, which is not wrapped with nanotechnology, can also recover to a certain extent at 20 minutes, reaching 50.51% of the initial blood flow, but the thrombolytic efficacy is significantly different from that of TNK-DNaseⅠ@ZPM bionic nanosystem (p<0.0001).

[0136] After administration, the occluded carotid arteries of the FeCl3 carotid artery thrombosis model mice in each group were isolated, and the residual area of ​​thrombus in the blood vessels was evaluated using HE staining. Except for the normal saline group, the thrombus area in the occluded carotid artery of each group of mice was reduced. Among them, the thrombus-dissolving effect of the TNK-DNaseⅠ@ZPM biomimetic nanosystem group was the best (TNK-DNaseⅠ@ZPM vs TNK+DNase-Ⅰ, p < 0.01; TNK-DNaseⅠ@ZPM vs the other groups, p < 0.0001). Although some filamentous or flaky thrombus tissue was still shown in the vascular section images, most of them were stained lightly, indicating that the TNK-DNaseⅠ@ZPM biomimetic nanosystem almost completely dissolved the thrombus in the carotid artery in a short period of time ( Figure 11 A~B in it).

[0137] In photochemical thromboembolism model mice, laser speckle was used to observe the recovery of cerebral blood flow in mice within 1 hour of drug administration. Figure 12 Figures A to B show the cerebral blood flow of different treatment groups at different time points (before modeling, 0, 20, 40, and 60 minutes after administration). Except for the saline group, the effects on cerebral blood flow in the other groups were different. The cerebral blood flow of mice in the TNK group recovered to a certain extent 60 minutes after administration, but the recovery was relatively small, reaching 31.72% of the baseline level. The cerebral blood flow of mice in the TNK+DNase-Ⅰ group recovered more significantly, recovering to 61.00% of the baseline level at 60 minutes, which was significantly higher than that of the TNK group (p < 0.0001). This shows that the combined use of TNK and DNase-Ⅰ has a better effect on the recovery of occluded cerebral blood flow.

[0138] The recovery of the TNK@ZIF-8 nanoparticle group and the TNK-DNaseⅠ@ZIF-8 nanoparticle group was relatively poor. The cerebral blood flow of these two groups of mice had not yet reached 30% of the baseline level 60 minutes after administration. This may be due to the small amount of drug released by the nanocarrier ZIF-8 in the circulation in a short period of time, resulting in a lower drug concentration at the thrombus site. Among all treatment groups, the TNK-DNaseⅠ@ZPM biomimetic nanosystem group had the most significant cerebral blood flow recovery effect, which recovered to 72.44% of the baseline level on average at 60 minutes (60 minutes, TNK-DNaseⅠ@ZPM vs TNK+DNase-Ⅰ, p < 0.001; TNK-DNaseⅠ@ZPM vs other groups, p < 0.0001). From the overall trend of cerebral blood flow recovery in the TNK-DNaseⅠ@ZPM biomimetic nanosystem group, the cerebral blood flow recovery was relatively slow in the initial stage. At 20 minutes, the effect was not as good as that of the TNK+DNase-Ⅰ group (20 minutes, TNK+DNase-Ⅰ vs TNK-DNaseⅠ@ZPM: 37.61% vs 31.90%, p < 0.01). At 40 minutes, there was no statistical difference in cerebral blood flow recovery between the two groups (40 minutes, TNK+DNase-Ⅰ vs TNK-DNaseⅠ@ZPM: 50.16% vs 49.48%, p=0.97). Until 60 minutes, the TNK-DNaseⅠ@ZPM biomimetic nanosystem group showed a better thrombolytic effect than the TNK+DNase-Ⅰ group, which shows that the sustained release of nanoparticles, due to its targeted nature, makes the drug accumulate more at the thrombus site, showing a superior therapeutic effect.

[0139] The Longa score was used to evaluate the neurological deficits in mice with photochemical thromboembolism 24 hours after the onset of the thromboembolism. The results showed that the neurological function of the TNK-DNaseⅠ@ZPM biomimetic nanosystem group was significantly improved compared to the normal saline and TNK@ZIF-8 nanoparticle groups (p < 0.01), and no significant statistical differences were found between the groups. TTC staining of brain tissue showed that the cerebral infarction volume of mice in each group decreased to varying degrees, but the cerebral infarction volume of mice in the TNK-DNaseⅠ@ZPM biomimetic nanosystem group was the smallest ( Figure 13 A to C in the figure), which was superior to TNK+DNase-Ⅰ and the other groups (TNK-DNaseⅠ@ZPM vs TNK+DNase-Ⅰ, p<0.001; TNK-DNaseⅠ@ZPM vs the other groups, p<0.0001).

[0140] Example 4 Thrombus targeting and organ distribution of TNK-DNase I@ZPM biomimetic nanosystem

[0141] 1. Experimental Methods

[0142] According to Example 3, a photochemical thromboembolism mouse model and a FeCl3 carotid artery thrombosis mouse model were constructed.

[0143] Indocyanine green-labeled TNK-DNase I@ZIF-8 nanoparticles or TNK-DNase I@ZPM biomimetic nanosystems were injected via the tail vein into mice modeling FeCl3 carotid artery thrombosis and photochemical thromboembolism. A small-animal in vivo near-infrared imaging system was used to record the location and intensity of fluorescence in the mice at different time points within 2 hours of administration. Fluorescence distribution and intensity in key organs of the mice were also observed 2 hours after administration.

[0144] 2. Experimental Results

[0145] In the FeCl3 carotid artery thrombosis model, the fluorescence signal of the TNK-DNaseⅠ@ZIF-8 nanoparticle group was found to be weak at all time points, and it was almost impossible to observe a clear fluorescent area, indicating that its accumulation in the neck area of ​​the mice was not obvious. In contrast, in the TNK-DNaseⅠ@ZPM biomimetic nanosystem group, a clear accumulation of fluorescent signals was observed in the neck area 3 minutes after administration. As time went on, the fluorescent signal remained strong and stable at 10 to 60 minutes, and the fluorescent area was also relatively concentrated. Then the signal weakened at 90 to 120 minutes. Figure 14 These results indicate that the TNK-DNaseⅠ@ZPM biomimetic nanosystem can rapidly accumulate at the thrombus site in mice in a short period of time and maintain a high concentration for a certain period of time before being gradually metabolized. The same results were shown in the photochemical thromboembolism model mice ( Figure 14 B) in.

[0146] In conclusion, in both animal models, the TNK-DNaseⅠ@ZPM biomimetic nanosystem showed better thrombus targeting than the uncoated TNK-DNaseⅠ@ZIF-8 nanoparticles ( Figure 14 C-D, 30-120 min, p < 0.0001). This difference may be related to the structural characteristics of the TNK-DNaseⅠ@ZPM biomimetic nanosystem. The platelet membrane wrapping gives it better targeting ability, enabling it to effectively aggregate at the thrombus site, thereby increasing the concentration of the drug at the lesion and enhancing the therapeutic efficacy.

[0147] In both animal models, 2 h after administration, TNK-DNaseⅠ@ZIF-8 nanoparticles and TNK-DNaseⅠ@ZPM biomimetic nanosystems showed strong fluorescence signals in the liver and kidney ( Figure 15Figures A–C indicate that the drug primarily accumulates in these two organs, with lower fluorescence intensity in the spleen, heart, lungs, and brain. These results suggest that TNK-DNaseⅠ@ZIF-8 nanoparticles or TNK-DNaseⅠ@ZPM biomimetic nanosystems may be primarily metabolized in the liver and kidneys, providing insights into the metabolic pathways and potential hepato-renal toxicity of these nanoparticles.

[0148] Example 5 Pharmacokinetics of TNK-DNase I@ZPM biomimetic nanosystem in vivo

[0149] 1. Experimental Methods

[0150] Indocyanine green-labeled TNK-DNaseⅠ@ZIF-8 nanoparticles or TNK-DNaseⅠ@ZPM biomimetic nanosystem (1 mg / mL) were injected into normal mice through the tail vein. Blood was collected from the mouse orbits at 1 min, 10 min, 30 min, 1 h, 4 h, 8 h, 24 h, and 48 h after injection. The blood fluorescence intensity was detected using a small animal in vivo near-infrared imaging system to evaluate the pharmacokinetics.

[0151] 2. Experimental Results

[0152] The results are as follows Figure 16 As shown in Figures A to B, a high fluorescence intensity appeared in the blood of mice in the TNK-DNaseⅠ@ZIF-8 nanoparticle group 1 minute after drug administration, indicating that the drug had quickly entered the blood circulation. As time went on, the fluorescence intensity gradually weakened. By 4 hours, the fluorescence intensity of the blood in the image was observed to have dropped significantly, indicating that the drug concentration in the circulation was significantly reduced. After 24 hours, the fluorescence in the blood almost disappeared, indicating that the metabolism or clearance rate of TNK-DNaseⅠ@ZIF-8 nanoparticles in the body was faster and the retention time was shorter.

[0153] The TNK-DNaseⅠ@ZPM biomimetic nanosystem also quickly enters the circulation in a short period of time. Unlike TNK-DNaseⅠ@ZIF-8 nanoparticles, a certain fluorescence intensity can still be observed within 4 to 24 hours of administration. Until 48 hours, the fluorescence signal in the blood becomes very weak, indicating that the encapsulated TNK-DNaseⅠ@ZPM biomimetic nanosystem circulates in mice for a longer time and has better sustainability and stability during drug delivery.

[0154] Example 6 Safety Evaluation of TNK-DNaseⅠ@ZPM Bionic Nanosystem

[0155] 1. Experimental Methods

[0156] A photochemical thromboembolism mouse model was constructed according to Example 3.

[0157] Physiological saline, TNK, and TNK-DNaseⅠ@ZPM biomimetic nanosystem were injected into photochemical thromboembolism model mice through the tail vein, respectively. Seven days later, whole blood was collected to detect biochemical indicators of the mice, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), uric acid (UA), urea (UREA), creatinine (CREA) and coagulation indicators, including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and fibrinogen (FIB). At the same time, the heart, liver, spleen, lung, and kidney were obtained for pathological analysis using HE staining.

[0158] 2. Experimental Results

[0159] HE staining results of important organs of mice with photochemical thromboembolism are as follows Figure 17 As shown in Figure A, from the results of tissue section observation, the tissue morphology of various organs in the TNK-DNaseⅠ@ZPM biomimetic nanosystem group was similar to that in the normal saline group and the TNK group, and no obvious tissue damage, inflammatory response or cytopathic changes were observed. This provides histological evidence that the TNK-DNaseⅠ@ZPM biomimetic nanosystem has good safety in vivo.

[0160] Analysis results of key blood indicators in mice with photochemical thromboembolism 7 days after administration Figure 17 As shown in Figure B, the results showed that there was no significant statistical difference in the biochemical indicators of mice including ALT, AST, UREA, CREA and UA among the normal saline group, TNK group and TNK-DNaseⅠ@ZPM bionic nanosystem group, which preliminarily indicated that the TNK-DNaseⅠ@ZPM bionic nanosystem did not cause obvious damage or functional changes in the liver and kidneys, although judging from the organ distribution in the body, the TNK-DNaseⅠ@ZPM bionic nanosystem may be mainly metabolized through the liver and kidneys.

[0161] The analysis results of coagulation indicators including PT, APTT, TT and FIB showed that the effect of TNK-DNaseⅠ@ZPM bionic nanosystem on the reduction of APTT was different from that of TNK (p < 0.05), but there was no significant difference compared with the normal saline group. The other coagulation indicators showed no statistical differences between the normal saline group and the TNK group. These results indicate that although the TNK-DNaseⅠ@ZPM bionic nanosystem has a slight effect on some coagulation indicators, its effect on coagulation is smaller than that of TNK, and the risk of bleeding caused by this may be relatively low.

[0162] Comparative Example 1 Effect of Water on the Preparation of DNase-Ⅰ@ZIF-8 Nanoparticles and TNK-DNaseⅠ@ZIF-8 Nanoparticles

[0163] 1. Experimental Methods

[0164] DNase-I@ZIF-8 nanoparticles and TNK-DNaseI@ZIF-8 nanoparticles were prepared according to the method of Example 1, except that Milli-Q ultrapure water was replaced with pure water during the preparation process. The zeta potential of the prepared nanoparticles was characterized using a Zetasizer Nano ZS particle analyzer.

[0165] 2. Experimental Results

[0166] When pure water is used as a solvent, since pure water contains metal ions, DNase-Ⅰ will react with metal ions to form a positive charge ( Figure 18 Solution 1) cannot be encapsulated into ZIF-8, which is also positively charged, resulting in the inability to successfully prepare DNase-Ⅰ@ZIF-8 nanoparticles and TNK-DNaseⅠ@ZIF-8 nanoparticles.

[0167] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A biomimetic nanosystem, characterized in that: The biomimetic nanosystem comprises an inner core and an outer membrane, wherein the inner core comprises tenecteplase, deoxyribonuclease I and an organic metal framework; The outer membrane wraps around the outer periphery of the inner core.

2. The biomimetic nanosystem according to claim 1, characterized in that The organic metal framework is a zeolite imidazolate framework.

3. The biomimetic nanosystem according to claim 1, characterized in that The outer membrane is the cell membrane.

4. The biomimetic nanosystem according to claim 3, characterized in that: The cell membrane includes a platelet membrane.

5. The method for preparing the biomimetic nanosystem according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. Thoroughly mix tenecteplase, deoxyribonuclease I, 2-methylimidazole, zinc salt, surfactant, and ultrapure water to prepare TNK-DNase I@ZIF-8 nanoparticles; S2. Add the outer membrane to the TNK-DNaseⅠ@ZIF-8 nanoparticle dispersion and mix thoroughly so that the outer membrane wraps the TNK-DNaseⅠ@ZIF-8 nanoparticles to prepare a biomimetic nanosystem.

6. The preparation method according to claim 5, characterized in that In step S1, the surfactant includes polyvinyl pyrrolidone.

7. The preparation method according to claim 5, characterized in that In step S1, the mass ratio of tenecteplase to deoxyribonuclease I is 2.5-3.5:0.25-0.

30.

8. The preparation method according to claim 5, characterized in that In step S2, the mass ratio of the TNK-DNaseⅠ@ZIF-8 nanoparticles to the outer membrane is 0.8-1.2:0.8-1.

2.

9. Use of the biomimetic nanosystem according to any one of claims 1 to 4 in the preparation of a drug for treating acute ischemic stroke.

10. A drug for treating acute ischemic stroke, characterized in that: The bionic nanosystem comprises any one of claims 1 to 4.

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