A thrombolytic-targeting nanocomplex and a preparation method and application thereof

Abstract

The application discloses a thrombolytic nano-complex, a preparation method and application thereof, and belongs to the technical field of biological medicines, and provides a thrombolytic nano-complex, wherein the thrombolytic nano-complex is a fluorescently modified ROS-responsive dendritic mesoporous silica, the pores of the dendritic mesoporous silica are loaded with ferroferric oxide and tissue-type plasminogen activator, and the surface of the dendritic mesoporous silica is connected with a CREKA polypeptide. The thrombolytic nano-complex is a fluorescently modified ROS-responsive dendritic mesoporous silica, the pores of the dendritic mesoporous silica are loaded with ferroferric oxide and tissue-type plasminogen activator, and the surface of the dendritic mesoporous silica is connected with a CREKA polypeptide, so that the side effects of the existing thrombolytic method, such as poor targeting, low specificity and high risk of causing bleeding, are solved, the drug aggregation and permeability are improved, it is a biological-targeting intelligent thrombolytic system, and a new thrombolytic direction integrating chemistry and biology is provided.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a targeted thrombolytic nanocomposite, its preparation method, and its application. Background Technology

[0002] Stroke, also known as cerebrovascular accident, is characterized by high incidence, high mortality, high disability rate, and high recurrence rate. It is a cerebrovascular disease caused by various triggering factors, resulting in narrowing, occlusion, or rupture of cerebral arteries. It is generally divided into two main categories: hemorrhagic stroke and ischemic stroke. Ischemic stroke is a disease caused by thrombosis or embolism, which interrupts blood flow to the brain, leading to ischemic and hypoxic damage to brain tissue. Its core mechanisms include the rupture of atherosclerotic plaques forming thrombi (cerebral thrombosis), or emboli from the heart or other parts of the body breaking off and blocking distal cerebral blood vessels (cerebral embolism). Exploring and improving emergency treatment strategies for ischemic stroke has always been a hot topic and a challenge in medical research. Currently, intravenous thrombolysis is one of the methods for treating ischemic stroke. However, clinical practice has shown that after intravenous injection of anticoagulants (also known as tissue plasminogen activator, tPA), the recanalization rate of the target vessel is low, and the low recanalization rate limits the long-term efficacy of this treatment.

[0003] The reasons for the difficulty in restoring blood flow in patients with ischemic stroke are as follows: First, during an ischemic stroke, the blockage of the affected blood vessel leads to an interruption of local blood flow, making it difficult for thrombolytic drugs to reach the vicinity of the thrombus through circulation. Second, thrombolytic drugs have short half-lives and are rapidly degraded after intravenous injection, failing to form a high concentration at the thrombus site. Therefore, improving the targeting of thrombolytic drugs is key to solving the problem of low blood flow restoration rates. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a targeted thrombolytic nanocomposite, its preparation method, and its application. This solves the side effects of existing thrombolytic methods, such as poor targeting, low specificity, and high risk of bleeding. The targeted thrombolytic nanocomposite is a fluorescently modified ROS-responsive dendritic mesoporous silica, with its pores loaded with iron(II,III) oxide and tissue plasminogen activator, and its surface linked with CREKA peptides, which improves drug aggregation and permeability. This provides a novel thrombolytic approach that integrates chemistry and biology for a biologically targeted intelligent thrombolytic system.

[0005] To achieve the above objectives, the present invention provides a targeted thrombolytic nanocomposite, wherein the targeted thrombolytic nanocomposite is a fluorescently modified ROS-responsive dendritic mesoporous silica, wherein the pores are loaded with iron oxide and tissue plasminogen activator, and the surface is connected with CREKA peptide.

[0006] Preferably, the fluorescently modified ROS-responsive dendritic mesoporous silicon is CY5.5-modified ROS-responsive dendritic mesoporous silicon.

[0007] Preferably, the targeted thrombolytic nanocomposite is spherical with a particle size of 230.403±0.88 nm.

[0008] Preferably, the electrokinetic potential of the targeted thrombolytic nanocomposite is -8.357±3.68mV.

[0009] The present invention also provides a method for preparing the targeted thrombolytic nanocomposite, comprising the following steps:

[0010] (1) Triethanolamine (TEA) is mixed with deionized water and stirred for the first time. Then hexadecyltrimethylammonium bromide (CTAB) and sodium salicylate (NaSal) are added and stirred for the second time to obtain a preliminary mixed solution.

[0011] (2) Add ethyl silicate (TEOs) and bis-[3-(triethoxysilyl)propyl]-disulfide to the initial mixed solution obtained in step (1), react, and then centrifuge to collect the precipitate to obtain the product. Calcine to obtain ROS-responsive dendritic mesoporous silica (MSNP).

[0012] (3) Mix the ROS-responsive dendritic mesoporous silica obtained in step (2) with the fluorescent dye solution, stir for the third time, and then centrifuge for the second time to collect the precipitate to obtain fluorescent modified ROS-responsive dendritic mesoporous silica.

[0013] (4) Mix iron oxide with aminopropyltriethoxysilane (APTES), stir, and then centrifuge to collect the precipitate to obtain amino-modified iron oxide.

[0014] (5) Mix the fluorescently modified ROS-responsive dendritic mesoporous silica, tissue plasminogen activator (TPA), CREKA peptide (CREKA) obtained in step (3) with the amino-modified iron oxide obtained in step (4), stir in the fifth step, and then centrifuge in the fourth step to collect the precipitate to obtain the targeted thrombolytic nanocomposite (tPA-SPION-MSNP-CREKA).

[0015] Preferably, the mixing ratio of triethanolamine to deionized water in step (1) is 1g:40mL; the stirring speed of the first stirring in step (1) is 500rpm, the stirring temperature is 80℃, and the stirring time is 0.5h; the ratio of triethanolamine, hexadecyltrimethylammonium bromide and sodium salicylate in step (1) is 1g:0.25g:0.2mmol; the stirring speed of the second stirring in step (1) is 800rpm, the stirring temperature is 30℃, and the stirring time is 1h.

[0016] Preferably, the ratio of ethyl silicate, bis-[3-(triethoxysilyl)propyl]-disulfide, and triethanolamine used in step (1) in step (2) is 2g:0.5mL:1g; the reaction temperature in step (2) is 40℃, and the reaction time is 3h; the temperature of the first centrifugation in step (2) is 4℃, the speed of the first centrifugation is 10000rpm, and the time of the first centrifugation is 10min; the calcination temperature in step (2) is 550℃, and the calcination time is 6h.

[0017] Preferably, in step (3), the ratio of the ROS-responsive dendritic mesoporous silica to the fluorescent dye solution is 5 mg: 500 μL, and the concentration of the fluorescent dye solution in step (3) is 0.02 mmol / mL; the speed of the third stirring in step (3) is 600 rpm, the temperature of the third stirring is 25°C, and the time of the third stirring is 2 h; the temperature of the second centrifugation in step (3) is 4°C, the speed of the second centrifugation is 12000 rpm, and the time of the second centrifugation is 10 min.

[0018] Preferably, the mixing ratio of iron(III) oxide and aminopropyltriethoxysilane in step (4) is 10 mg: 100 μL; the stirring speed of the fourth stirring in step (4) is 800 rpm, the stirring temperature is 70°C, and the stirring time is 3 h; the centrifugation temperature of the third centrifugation in step (4) is 4°C, the centrifugation speed is 12000 rpm, and the centrifugation time is 10 min; the mixing ratio of the fluorescently modified ROS-responsive dendritic mesoporous silica, tissue plasminogen activator, CREKA peptide obtained in step (3), and the amino-modified iron(III) oxide obtained in step (4) in step (5) is 5 mg: 100 μg: 0.01 mmol: 5 mg; the stirring speed of the fifth stirring in step (5) is 600 rpm, the stirring temperature is 4°C, and the stirring time is 2 h; the centrifugation temperature of the fourth centrifugation in step (5) is 4°C, the centrifugation speed is 12000 rpm, and the centrifugation time is 10 min.

[0019] This invention also provides the application of the targeted thrombolytic nanocomposite in the preparation of drugs for treating ischemic stroke.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] This invention provides a targeted thrombolytic nanocomposite that modifies ROS-responsive dendritic mesoporous silica with the targeting peptide CREKA (Cys-Arg-Glu-Lys-Ala), which specifically recognizes thrombus fibrin. Leveraging the high specific surface area and strong loading capacity of mesoporous silica, the thrombolytic drug TPA and superparamagnetic iron oxide are simultaneously loaded into its mesopores. This design significantly increases the local TPA concentration at the thrombus, prolongs the plasma half-life of the thrombolytic drug, improves the safe dosage of the thrombolytic drug, and reduces the risk of post-thrombolysis bleeding. Simultaneously, due to the magnetic effect of the superparamagnetic iron oxide, the drug's permeability to the thrombus is improved, thereby improving the prognosis of ischemic stroke.

[0022] The targeted thrombolytic nanocomposite of this invention has a strong loading capacity. Utilizing the high specific surface area and porous structure of mesoporous silica, it can accommodate a large number of drug molecules. It has the function of rapidly targeting thrombi. ROS-responsive dendritic mesoporous silica modified with CREKA utilizes the specific binding of CREKA to fibrin in the thrombus to increase the aggregation rate of thrombolytic drugs around the thrombus. It reduces the peripheral degradation of thrombolytic drugs. The narrow mesopores of ROS-responsive dendritic mesoporous silica restrict the structural development of protein phospholipids (TPA), reducing the degradation of TPA in the periphery. It has controllable release characteristics. When the drug reaches the vicinity of the thrombus, the drug molecules are slowly released by stimulating the alternating magnetic field, exhibiting low initial burst release and sustained sustained release characteristics, which makes the drug release more stable and prolonged. It has a synergistic thrombolytic effect. The ROS-responsive dendritic mesoporous silica loaded with TPA and the CY5.5 surface modified with iron oxide, combined with alternating magnetic field stimulation, can enhance the release of TPA. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a schematic diagram illustrating the synthesis process of the targeted thrombolytic nanocomposite tPA-SPION-MSNP-CREKA of the present invention;

[0025] Figure 2 Particle size distribution and potential diagrams of MSNP prepared in Example 1, tPA-SPION-MSNP prepared in Comparative Example 1, and tPA-SPION-MSNP-CREKA prepared in Example 1 are shown, where A is the particle size distribution diagram and B is the potential diagram.

[0026] Figure 3Scanning electron microscope (SEM) and transmission electron microscope (TEM) images of the MSNP prepared in Example 1, the tPA-SPION-MSNP prepared in Comparative Example 1, and the tPA-SPION-MSNP-CREKA prepared in Example 1 are shown. In the images, A is a TEM image of the MSNP prepared in Example 1, B is a TEM image of the tPA-SPION-MSNP prepared in Comparative Example 1, C is a TEM image of the tPA-SPION-MSNP-CREKA prepared in Example 1, D is a SEM image of the MSNP prepared in Example 1, E is a SEM image of the tPA-SPION-MSNP prepared in Comparative Example 1, and F is a SEM image of the tPA-SPION-MSNP-CREKA prepared in Example 1. The scale bar is 200 nm.

[0027] Figure 4 The results of the fibrinolysis plate experiment for tPA-SPION-MSNP prepared in Comparative Example 1 and tPA-SPION-MSNP-CREKA prepared in Example 1 are shown. In the figure, A is a physical image of the fibrinolysis plate experiment, B is a quantitative statistical graph of the diameter of the transparent zone, and "*" in the figure represents the analysis of significant difference. C is an image of the binding of fluorescently labeled nanoparticles on the fibrin gel plate, and D is a statistical graph of the average fluorescence intensity of the fluorescence image. "*" in the figure represents the analysis of significant difference.

[0028] Figure 5 The results of in vitro thrombolytic activity assays for tPA-SPION-MSNP prepared in Comparative Example 1 and tPA-SPION-MSNP-CREKA prepared in Example 1 are shown. In the figures, A is a comparison of thrombi after 6 hours of treatment and thrombi before thrombolysis in each group; B is the percentage of thrombolysis after 6 hours of treatment in each group; "*" and "**" in the figures represent significant differences; C is an observation of fibrin release from the supernatant during 6 hours of thrombus treatment in each group; and D is a statistical chart of fibrin content released from the supernatant during 6 hours of thrombus treatment in each group; "*", "**", and p = 0.7 in the figures represent significant differences.

[0029] Figure 6 The in vivo thrombolytic capacity test results of tPA-SPION-MSNP prepared in Comparative Example 1 and tPA-SPION-MSNP-CREKA prepared in Example 1 are shown in A, which is the cerebral blood flow perfusion map (LSCI) of mice in each group, and B is the difference in cerebral infarction volume shown by TTC staining of the brain of mice in each group.

[0030] Figure 7The results of the in vivo thrombolytic ability analysis of tPA-SPION-MSNP prepared in Comparative Example 1 and tPA-SPION-MSNP-CREKA prepared in Example 1 are shown. In the figure, A shows the trend of cerebral blood flow perfusion (CBF) at different time points after surgery in different treatment groups, and B shows the comparison of the percentage of cerebral infarction volume in mice in different treatment groups. "*" in the figure represents the significance analysis of the difference. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] The materials used in this invention were sourced as follows: triethanolamine was purchased from Sinopharm Chemical Reagent Co., Ltd.; hexadecyltrimethylammonium bromide was purchased from Aladdin Reagent Co., Ltd.; sodium salicylate was purchased from Sinopharm Chemical Reagent Co., Ltd.; ethyl silicate was purchased from Aladdin Reagent Co., Ltd.; bis-[3-(triethoxysilyl)propyl]-disulfide was purchased from Xi'an Ruixi; CY5.5 was purchased from Beyotime Biotechnology Co., Ltd.; iron tetroxide was purchased from Xi'an Ruixi; aminopropyltriethoxysilane was purchased from Aladdin Reagent Co., Ltd.; tissue plasminogen activator was purchased from Boehringer Ingelheim; and CREKA peptide was purchased from Xi'an Ruixi.

[0037] Example 1

[0038] (1) Triethanolamine (TEA) and deionized water were mixed at a ratio of 1 g: 40 mL, and stirred at 500 rpm and 80 °C for 0.5 h. Then, hexadecyltrimethylammonium bromide (CTAB) and sodium salicylate (NaSal) were added, and stirred at 800 rpm and 30 °C for 1 h to obtain a preliminary mixed solution. The ratio of triethanolamine, hexadecyltrimethylammonium bromide and sodium salicylate was 1 g: 0.25 g: 0.2 mmol / L.

[0039] (2) Tetraethyl silicate (TEOs) and bis-[3-(triethoxysilyl)propyl]-disulfide were added to the initial mixed solution and reacted at 40°C for 3 h. Then, the mixture was centrifuged at 10,000 rpm for 10 min at 4°C. The precipitate was collected and washed three times alternately with deionized water and ethanol to remove residual reactants, yielding the product. The product was then calcined at 550°C for 6 h to remove the structure-directing agent, yielding ROS-responsive dendritic mesoporous silica (MSNP). The ratio of tetraethyl silicate, bis-[3-(triethoxysilyl)propyl]-disulfide, and triethanolamine used was 2 g: 0.5 mL: 1 g.

[0040] (3) ROS-responsive dendritic mesoporous silica was mixed with CY5.5 solution at a ratio of 5 mg: 500 μL. The concentration of CY5.5 solution was 0.02 mmol / mL. The mixture was stirred for 2 h at 25 °C and 600 rpm for the third time. Then it was centrifuged for 10 min at 4 °C and 12000 rpm for the second time to remove unloaded CY5.5. The precipitate was then collected to obtain fluorescently modified ROS-responsive dendritic mesoporous silica.

[0041] (4) Iron oxide and aminopropyltriethoxysilane (APTES) were mixed at a ratio of 10 mg: 100 μL, stirred at 70 °C and 800 rpm for 3 h, and then centrifuged at 4 °C and 12000 rpm for 10 min. The precipitate was collected and washed with deionized water to obtain amino-modified iron oxide.

[0042] (5) Fluorescently modified ROS-responsive dendritic mesoporous silica, tissue plasminogen activator (TPA), CREKA peptide (CREKA) and amino-modified iron oxide were mixed in a ratio of 5 mg: 100 μg: 0.01 mmol: 5 mg, stirred at 4 °C and 600 rpm for 2 h, and then centrifuged at 4 °C and 12000 rpm for 10 min. The precipitate was collected and washed with deionized water to obtain the targeted thrombolytic nanocomposite (tPA-SPION-MSNP-CREKA).

[0043] like Figure 1 The diagram shown illustrates the synthesis process of the targeted thrombolytic nanocomposite (tPA-SPION-MSNP-CREKA).

[0044] Comparative Example 1

[0045] (1) Triethanolamine (TEA) and deionized water were mixed at a ratio of 1 g: 40 mL, and stirred at 500 rpm and 80 °C for 0.5 h. Then, hexadecyltrimethylammonium bromide (CTAB) and sodium salicylate (NaSal) were added, and stirred at 800 rpm and 30 °C for 1 h to obtain a preliminary mixed solution. The ratio of triethanolamine, hexadecyltrimethylammonium bromide and sodium salicylate was 1 g: 0.25 g: 0.2 mmol / L.

[0046] (2) Tetraethyl silicate (TEOs) and bis-[3-(triethoxysilyl)propyl]-disulfide were added to the initial mixed solution and reacted at 40°C for 3 h. Then, the mixture was centrifuged at 10,000 rpm for 10 min at 4°C. The precipitate was collected and washed three times alternately with deionized water and ethanol to remove residual reactants, yielding the product. The product was calcined at 550°C for 6 h to remove the structure-directing agent, yielding ROS-responsive dendritic mesoporous silica (MSNP). The ratio of tetraethyl silicate, bis-[3-(triethoxysilyl)propyl]-disulfide, and triethanolamine used was 2 g: 0.5 mL: 1 g.

[0047] (3) ROS-responsive dendritic mesoporous silica was mixed with CY5.5 solution at a ratio of 5 mg: 500 μL. The concentration of CY5.5 solution was 0.02 mmol / mL. The mixture was stirred for 2 h at 25 °C and 600 rpm for the third time. Then it was centrifuged for 10 min at 4 °C and 12000 rpm for the second time to remove unloaded CY5.5. The precipitate was then collected to obtain fluorescently modified ROS-responsive dendritic mesoporous silica.

[0048] (4) Iron oxide and aminopropyltriethoxysilane (APTES) were mixed at a ratio of 10 mg: 100 μL, stirred at 70 °C and 800 rpm for 3 h, and then centrifuged at 4 °C and 12000 rpm for 10 min. The precipitate was collected and washed with deionized water to obtain amino-modified iron oxide.

[0049] (5) Fluorescently modified ROS-responsive dendritic mesoporous silica, tissue plasminogen activator (TPA) and amino-modified iron oxide were mixed in a ratio of 5 mg: 100 μg: 5 mg, stirred at 4 °C and 600 rpm for 2 h, and then centrifuged at 4 °C and 12000 rpm for 10 min. The precipitate was collected and washed with deionized water to obtain the targeted thrombolytic nanocomposite (tPA-SPION-MSNP).

[0050] Experimental Example 1

[0051] The particle size and potential of MSNP, tPA-SPION-MSNP, and tPA-SPION-MSNP-CREKA were determined using a nanoparticle size potentiometer (NanoBrook 90plus PALS). MSNP, tPA-SPION-MSNP, and tPA-SPION-MSNP-CREKA were characterized by TEM and SEM.

[0052] like Figure 2 As shown in Figure A, the particle sizes of MSNP, tPA-SPION-MSNP, and tPA-SPION-MSNP-CREKA are 232.433±2.35 nm, 232.023±8.55 nm, and 230.403±0.88 nm, respectively, indicating that the introduction of the polypeptide chain did not significantly change the particle size of the nanoparticles. Figure 2 As shown in Figure B, the electrokinetic potentials of MSNP, tPA-SPION-MSNP, and tPA-SPION-MSNP-CREKA are -10.77±0.21mV, -13.023±0.71mV, and -8.357±3.68mV, respectively.

[0053] like Figure 3 As shown, Figure 3 China A Figure 3 China B and Figure 3 C represents the TEM images of MSNP, tPA-SPION-MSNP, and tPA-SPION-MSNP-CREKA, respectively, as shown in the figure. Figure 3 Image A shows a transmission electron microscope (TEM) image of blank mesoporous silica nanoparticles (MSNPs). They exhibit a regular spherical morphology with a typical dendritic radial pore structure on the surface, uniformly distributed, and a particle size of approximately 200 nm. Figure 3 Image B represents the MSNP (tPA-SPION-MSNP) loaded with superparamagnetic iron oxide nanoparticles (SPION) and tissue plasminogen activator (tPA). Black, high-electron-density regions can be observed on some particle surfaces, representing the SPION loading signal. Figure 3The image shows nanoparticles tPA-SPION-MSNP-CREKA after further modification with the CREKA peptide. Compared to tPA-SPION-MSNP, tPA-SPION-MSNP exhibits more black, clustered, high-density regions on its surface, indicating that the CREKA peptide has been successfully modified onto the support surface. Simultaneously, some nanoparticle channels appear blurred, suggesting that the CREKA modification has a certain impact on the local structure, potentially enhancing the compactness and stability of the support structure. Figure 3 D, Figure 3 China E and Figure 3 F represents the SEM images of MSNP, tPA-SPION-MSNP, and tPA-SPION-MSNP-CREKA, respectively. From the SEM images, it can be directly observed that the MSNP prepared in Example 1, the tPA-SPION-MSNP prepared in Comparative Example 1, and the tPA-SPION-MSNP-CREKA prepared in Example 1 contain a large number of pores on their surfaces, and the pores on the surface of tPA-SPION-MSNP-CREKA have disappeared, indicating that the modification of CREKA peptides improves the stability of the nano-ionic structure.

[0054] Experiment Example 2

[0055] Biological targeting and in vitro fibrinolytic capacity assay:

[0056] To verify whether the targeted thrombolytic nanocomposite has a biotargeting effect on thrombi, a fibrin gel containing fibrinogen and thrombin was first prepared at 37°C. Equidistant wells were punched into the fully coagulated fibrin gel using a 3mm needle, leaving 6 test wells per plate. TPA, an equal volume of tPA-SPION-MSNP-CREKA prepared in Example 1, or an equal volume of tPA-SPION-MSNP prepared in Comparative Example 1 were then added, and the plates were incubated at 37°C for 30 min to ensure sufficient binding with fibrin. After incubation, the plates were gently washed three times with 1 mL PBS to remove non-specifically adsorbed particles. The fibrinolytic activity of different drugs was compared by measuring the diameter of the fibrinolytic zone. Three random fields of view were acquired from each plate under the same light source and exposure parameters using a fluorescence microscope (λ_ex = 488 nm). The total fluorescence integral density was quantified using ImageJ. The fibrinolytic activity of different drugs was compared by measuring the diameter of the fibrinolytic zone.

[0057] like Figure 4 China A Figure 4 B, Figure 4 C and Figure 4As shown in Figure D, the transparency aperture of tPA-SPION-MSNP-CREKA is twice that of tPA-SPION-MSNP, and the average fluorescence intensity of tPA-SPION-MSNP-CREKA on the fiber plate is about 1.5 times that of tPA-SPION-MSNP. The difference is statistically significant (P<0.05, n=3 fields of view).

[0058] The diameter of the transparent aperture is as follows Figure 4 China A and Figure 4 As shown in Figure B, the diameters of the clear zones formed by tPA and tPA-SPION-MSNP are similar (≈0.6 cm), indicating that the thrombolytic activity of tPA was not significantly reduced after encapsulation. The diameter of the clear zone formed by tPA-SPION-MSNP-CREKA was significantly increased to approximately twice that of tPA-SPION-MSNP (P<0.05, n=3), indicating that under the same enzyme activity conditions, CREKA modification significantly enhanced the local fibrinolytic ability of tPA. Fluorescence binding experiments are shown below. Figure 4 C and Figure 4 As shown in Figure D, the average fluorescence intensity of tPA-SPION-MSNP-CREKA on the fibrin mesh was approximately 1.5 times that of tPA-SPION-MSNP (P<0.05, n=3 fields of view), indicating that the CREKA peptide significantly improved the specific adsorption of the nanocarrier to the fibrin mesh. Combined with clear zone and quantitative fluorescence assays, it was demonstrated that CREKA could efficiently enrich tPA-SPION-MSNP on the fibrin mesh surface, increasing local enzyme concentration and amplifying the fibrinolytic effect at the same dosage. The results indicate that the CREKA peptide significantly improved the specific binding ability of TPA to the fibrin mesh, validating its superior biotargeting ability.

[0059] Experimental Example 3

[0060] In vitro thrombolytic capacity test:

[0061] 120 mg of pre-prepared fresh mouse thrombi were placed into EP tubes containing 1 mL of physiological saline, TPA (0.2 mg / mL), tPA-SPION-MSNP (free TPA concentration 0.2 mg / mL) prepared in Comparative Example 1, and tPA-SPION-MSNP-CREKA (free TPA concentration 0.2 mg / mL) prepared in Example 1, and incubated in a water bath at 37°C. The thrombus mass was measured after 6 hours of treatment and compared with the thrombus mass before thrombolysis to calculate the thrombolysis percentage. The absorbance (OD) of the supernatant at 410 nm at different time points was measured to calculate the fibrin release in the thrombus, thereby analyzing the thrombolytic effect.

[0062] like Figure 5 China A and Figure 5As shown in Figure B, the percentage of thrombus dissolution was calculated by comparing the thrombus mass 6 hours after treatment with the thrombus mass before thrombolysis. It is visually apparent that over time, the thrombus volume decreases and becomes thinner, and the supernatant of the solution changes from colorless to dark red. Figure 5 C and Figure 5 As shown in Figure D, the study found that starting at 4 hours of treatment, the tPA-SPION-MSNP-CREKA group released significantly more fibrin from the supernatant than the tPA-SPION-MSNP group. Furthermore, after 6 hours of treatment, the thrombi became significantly thinner and softer, and the thrombolysis percentage in the tPA-SPION-MSNP-CREKA group was significantly higher than that in the tPA-SPION-MSNP group. These results suggest that tPA-SPION-MSNP-CREKA treatment can improve thrombolytic efficacy.

[0063] Experiment Example 4

[0064] In vivo thrombolytic capacity test:

[0065] To investigate the in vivo thrombolytic activity of tPA-SPION-MSNP-CREKA prepared in Example 1, it was evaluated in a mouse model of MCAO. Mice were randomly divided into four groups, each treated with equal volumes of saline, TPA, tPA-SPION-MSNP prepared in Comparative Example 1, and tPA-SPION-MSNP-CREKA prepared in Example 1, respectively. Taking tPA-SPION-MSNP-CREKA as an example, after successful treatment confirmed by laser speckle flow imaging 10 minutes post-surgery, tPA-SPION-MSNP-CREKA (1 mg tPA·kg⁻¹) was injected via the tail vein. -1 The dosage was calculated based on tPA equivalents, with tPA content in the formulation: 5mg carrier ≈ 100μg tPA. A magnet was used to guide tPA-SPION-MSNP-CREKA to the M1 segment of the middle cerebral artery, i.e., the thrombus site. Local cerebral blood flow perfusion was monitored preoperatively, at 0h postoperatively, 3h postoperatively, and 6h postoperatively, and changes in cerebral blood flow were compared among the four groups.

[0066] The results are as follows Figure 6 China A Figure 6 B, Figure 7 China A and Figure 7 As shown in Figure B, the study found that cerebral blood flow significantly recovered 6 hours after surgery with tPA-SPION-MSNP-CREKA. Neurological deficits were assessed using mNSS at 6 hours post-surgery, and infarct volume was compared using TTC. The infarct volume in the tPA-SPION-MSNP-CREKA group was smaller than that in the tPA-SPION-MSNP group. These findings demonstrate, at the in vivo level, that the synergistic effect of CREKA improves neurological function.

[0067] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A targeted thrombolytic nanocomposite, characterized in that, The targeted thrombolytic nanocomposite is a fluorescently modified ROS-responsive dendritic mesoporous silica, in which iron oxide and tissue plasminogen activator are loaded in the pores, and CREKA peptide is attached to the surface. The targeted thrombolytic nanocomposite was prepared by mixing 5 mg of fluorescently modified ROS-responsive dendritic mesoporous silica, 100 μg of tissue plasminogen activator, 0.01 mmol of CREKA peptide, and 5 mg of amino-modified iron oxide.

2. The targeted thrombolytic nanocomposite according to claim 1, characterized in that, The fluorescently modified ROS-responsive dendritic mesoporous silicon is CY5.5-modified ROS-responsive dendritic mesoporous silicon.

3. The targeted thrombolytic nanocomposite according to claim 1, characterized in that, The targeted thrombolytic nanocomposite is spherical with a particle size of 230.403±0.88 nm.

4. The targeted thrombolytic nanocomposite according to claim 1, characterized in that, The electrokinetic potential of the targeted thrombolytic nanocomposite is -8.357±3.68mV.

5. The method for preparing the targeted thrombolytic nanocomposite according to any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Triethanolamine is mixed with deionized water, stirred for the first time, then hexadecyltrimethylammonium bromide and sodium salicylate are added, stirred for the second time, and a preliminary mixed solution is obtained; (2) Add ethyl silicate and bis-[3-(triethoxysilane)propyl]-disulfide to the initial mixed solution obtained in step (1), react, and then centrifuge to collect the precipitate to obtain the product. Calcine to obtain ROS-responsive dendritic mesoporous silica. (3) Mix the ROS-responsive dendritic mesoporous silica obtained in step (2) with the fluorescent dye solution, stir for the third time, and then centrifuge for the second time to collect the precipitate to obtain fluorescent modified ROS-responsive dendritic mesoporous silica. (4) Mix iron oxide with aminopropyltriethoxysilane, stir, and then centrifuge to collect the precipitate to obtain amino-modified iron oxide. (5) Mix the fluorescently modified ROS-responsive dendritic mesoporous silica, tissue plasminogen activator, CREKA peptide and amino-modified iron oxide obtained in step (3), stir for the fifth time, and then centrifuge for the fourth time to obtain the precipitate, thus obtaining the targeted thrombolytic nanocomposite.

6. The preparation method according to claim 5, characterized in that, The mixing ratio of triethanolamine to deionized water in step (1) is 1g:40mL; the stirring speed of the first stirring in step (1) is 500rpm, the stirring temperature is 80℃, and the stirring time is 0.5h; the amount of triethanolamine added in step (1) is 1g, the amount of hexadecyltrimethylammonium bromide added is 0.25g, and the amount of sodium salicylate added is 0.2mmol; the stirring speed of the second stirring in step (1) is 800rpm, the stirring temperature is 30℃, and the stirring time is 1h.

7. The preparation method according to claim 5, characterized in that, The ratio of ethyl silicate, bis-[3-(triethoxysilyl)propyl]-disulfide, and triethanolamine used in step (1) in step (2) is 2g:0.5mL:1g; the reaction temperature in step (2) is 40℃, and the reaction time is 3h; the temperature of the first centrifugation in step (2) is 4℃, the speed of the first centrifugation is 10000rpm, and the time of the first centrifugation is 10min; the calcination temperature in step (2) is 550℃, and the calcination time is 6h.

8. The preparation method according to claim 5, characterized in that, In step (3), the ratio of the ROS-responsive dendritic mesoporous silica to the fluorescent dye solution is 5 mg: 500 μL, and the concentration of the fluorescent dye solution in step (3) is 0.02 mmol / mL; the speed of the third stirring in step (3) is 600 rpm, the temperature of the third stirring is 25°C, and the time of the third stirring is 2 h; the temperature of the second centrifugation in step (3) is 4°C, the speed of the second centrifugation is 12000 rpm, and the time of the second centrifugation is 10 min.

9. The preparation method according to claim 5, characterized in that, In step (4), the mixing ratio of iron(III) oxide and aminopropyltriethoxysilane is 10 mg: 100 μL; in step (4), the speed of the fourth stirring is 800 rpm, the temperature of the fourth stirring is 70 °C, and the time of the fourth stirring is 3 h; in step (4), the temperature of the third centrifugation is 4 °C, the speed of the third centrifugation is 12000 rpm, and the time of the third centrifugation is 10 min; in step (5), 5 mg of fluorescently modified ROS-responsive dendritic mesoporous silica, 100 μg of tissue plasminogen activator, 0.01 mmol of CREKA peptide and 5 mg of amino-modified iron(III) oxide obtained in step (4) are mixed; in step (5), the speed of the fifth stirring is 600 rpm, the temperature of the fifth stirring is 4 °C, and the time of the fifth stirring is 2 h; in step (5), the temperature of the fourth centrifugation is 4 °C, the speed of the fourth centrifugation is 12000 rpm, and the time of the fourth centrifugation is 10 min.

10. The use of the targeted thrombolytic nanocomposite as described in any one of claims 1 to 4 in the preparation of a drug for treating ischemic stroke.

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