A nano-MOF with pH-responsive function for targeted dual-drug delivery thrombolysis and its preparation method

Abstract

The present invention provides a nanoscale MOF with pH-responsive function for targeted dual-drug thrombolysis and a preparation method thereof. Hollow mineralized nanoscale calcium carbonate powder is mixed with γ-cyclodextrin and a potassium ion compound, ultrasonically dispersed, and then incubated to form a cyclodextrin MOF embedded with calcium carbonate. An RGD polypeptide is grafted onto the surface of the cyclodextrin MOF embedded with calcium carbonate to obtain MOF-RGD. The grafted MOF-RGD is impregnated into ethanol containing an anticoagulant drug, air-dried, and then mixed with a thrombolytic drug, so that the thrombolytic drug is loaded on the surface of the nanoscale MOF to obtain a pH-responsive targeted nanoscale MOF. The present invention forms a nanoscale MOF with a targeted function, which combines thrombolysis and anticoagulation, and realizes the treatment and prevention of deep vein thrombosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to a nano-MOF with pH-responsive function for targeted dual-drug thrombolysis and a preparation method thereof. Background Art

[0002] Although significant progress has been made in the treatment of cardiovascular and cerebrovascular diseases, they remain the main culprits of high incidence and high mortality in both developed and developing countries. Vascular embolism has become the leading cause of death from cardiovascular and cerebrovascular diseases. Once a thrombus is formed, it can lead to blockage or absolute occlusion of the blood circulation system, resulting in critical events such as ischemic stroke, acute myocardial infarction, and pulmonary embolism. Venous thromboembolism is a typical chronic disease among those induced by vascular embolism, affecting nearly 10 million people globally each year. Approximately 30% of patients with venous thromboembolism usually die within 30 days after the occurrence of pulmonary embolism. One-third to one-half of patients with venous thromboembolism have recurrent VT or long-term debilitating diseases, including chronic pain, edema, and stubborn venous leg ulcers (post-thrombotic syndrome). Increasing evidence indicates a complex interaction between coagulation and inflammation in venous thrombosis. The activation of the coagulation cascade triggers the immune system, and in turn, innate immune cells promote thrombus formation, a process known as immunothrombosis. Activated white blood cells and platelets are the main sources of procoagulant factors, which may stimulate the formation and growth of thrombi. Therefore, more than one-third of patients with DVT may develop chronic venous insufficiency, leading to leg pain, swelling, varicose veins, and venous ulcers, with few treatment methods. Therefore, it is urgent to develop a nano-material that integrates thrombolysis, anticoagulation, anti-inflammation, targeting, etc. according to the pathogenesis characteristics of venous thrombosis to meet the safety and effectiveness requirements of clinical surgery and postoperative prevention and treatment. This is a long-standing and urgently needed practical problem in the research and development field of thrombolytic nanomedicine.

[0003] To achieve good treatment effects for venous thrombosis, thrombolysis and anticoagulation are the main strategies against venous thromboembolism, and the first-line treatment with plasminogen activators (PAs) has been used as the main method for thrombolysis. However, the systemic administration of PAs is limited clinically by several factors, including their rapid neutralization by endogenous inhibitors (such as PA inhibitor-1) and the high risk of severe side effects (such as hemorrhagic complications), resulting in reduced treatment efficacy and a lower risk-benefit relationship. Among the most common PAs, urokinase PA (uPA) is inexpensive and widely used in developing countries to treat acute ischemic stroke. Although effective, its use is associated with a high risk of intracranial hemorrhage due to the lack of fibrin specificity. Studies have shown that the treatment with the non-steroidal anti-inflammatory drug aspirin is a potential alternative for the long-term secondary prevention of venous thromboembolism and can be used to extend oral anticoagulant therapy. The side effects of aspirin cannot be underestimated. As an oral anticoagulant, it has strong antiplatelet activity, and the main drug ingredient acetylsalicylic acid has a certain damaging effect on the gastric mucosa and poor solubility in the physiological environment. Therefore, the long-term abuse of the drug will cause various side effects such as systemic bleeding and coagulation dysfunction in VET patients. Moreover, the drug has no targeting property, is expensive, has a short half-life, is often limited to clinical use and has a high cost, and patients have to endure the torture of thromboembolic sequelae for a long time.

[0004] In recent years, many studies on new thrombolytic materials for venous thrombosis have been carried out at home and abroad. The research reports on a large number of thrombolytic materials and their thrombolytic mechanisms have promoted the rapid development of new thrombolytic materials in clinical applications. Most of them use the pathological properties of thrombus as a responsive release switch, use the cavity properties of nanoparticles to encapsulate PA to prevent leakage, and use photothermal magnetism to prevent re-blockage of thrombus and the onset of inflammation. Shuting Li et al. proposed a covalent organic framework engineered melanin nanoplateform based on platelet-mimicking porphyrin, aiming to target hirudin to the venous thrombus site to achieve non-invasive thrombolysis and effective anticoagulation. Thrombolysis is activated by hyperthermia and reactive oxygen species under near-infrared light irradiation. Jiasheng Xu et al. used a binary eutectic phase change fatty acid composed of lauric acid and stearic acid to block the pores of gold mesoporous silica core-shell nanoparticles, thereby providing thrombolytic drugs. The eutectic mixture has a definite melting point at 39.2 °C and can be used as a biocompatible phase change material for thermally triggered drug release. Local hyperthermia can enhance thrombus dissolution. Jinrong Zheng et al. used neutrophils as a natural carrier for thrombolysis to actively target the thrombus site enriched with inflammation, and inflammatory cytokines can activate UM-NEs (Ag-UK) to release NETs at the thrombus formation site, resulting in the simultaneous release of urokinase-loaded gold nanoparticles. Most of the current studies mainly use light (near-infrared), local hyperthermia as responsive release switches and the second factor to enhance deep vein thrombolysis and anticoagulation, or achieve the effect of multi-stage drug release. However, the biocompatibility and in vivo metabolism of gold-silver or porphyrin nanomaterials are still unsolved problems. Therefore, finding a new type of nano-targeted material that can release drugs in multiple stages and uses the pathological properties of thrombus as a responsive switch, and has high biosafety and low cost has become the main research direction for the treatment of deep vein thrombosis. Summary of the Invention

[0005] In order to solve the problems in the prior art, the present invention uses cyclodextrin MOF as the main carrier for drug delivery to form a nano-MOF with a targeting function that combines thrombolysis and anticoagulation, realizing the treatment and prevention of deep vein thrombosis, and providing a pH-responsive targeted dual-drug delivery thrombolytic nano-MOF and its preparation method.

[0006] The present invention realizes the solution of its technical problems by adopting the following technical solutions:

[0007] The first object of the present invention is to provide a pH-responsive targeted dual-drug delivery thrombolytic nano-MOF, which is characterized in that it includes a MOF wrapped with pH-responsive nanoparticles, and also includes RGD, a thrombolytic drug, and an anticoagulant drug.

[0008] Further, the thrombolytic drug is urokinase, lumbrokinase, streptokinase, nattokinase or tissue plasminogen activator. The anticoagulant drug is aspirin, dipyridamole, ticlopidine, clopidogrel, cilostazol, ozagrel, sarpogrelate, indobufen, prostacyclin, iloprost, dazoxiben, clofibrate, dextran 70, troxerutin, eptifibatide, tirofiban, abciximab, anagrelide or sulfinpyrazone.

[0009] Further, the nano-MOF with pH-responsive function for targeted dual-drug delivery thrombolysis includes cyclodextrin MOF, hollow mineralized nano-calcium carbonate embedded in the cyclodextrin MOF, RGD with targeting effect, anticoagulant drug loaded in the cavity and pores of the cyclodextrin MOF, and thrombolytic drug loaded on the surface of the MOF.

[0010] Further, the particle size of the nano-MOF is 200 - 300 nm.

[0011] The second object of the present invention is to provide a preparation method of a nano-MOF with pH-responsive function for targeted dual-drug delivery thrombolysis, which is characterized in that the hollow mineralized nano-calcium carbonate powder is mixed with γ-cyclodextrin and potassium ion compound, ultrasonically dispersed and then incubated to form cyclodextrin MOF embedded with calcium carbonate; RGD polypeptide is grafted on the surface of the cyclodextrin MOF embedded with calcium carbonate to obtain MOF-RGD; the grafted MOF-RGD is impregnated into ethanol dissolved with anticoagulant drug, air-dried and then mixed with thrombolytic drug to load the thrombolytic drug on the surface of the nano-MOF to obtain a pH-responsive targeted nano-MOF.

[0012] Further, the preparation method of the hollow mineralized nano-calcium carbonate is: dissolving calcium chloride dihydrate in absolute ethanol, adding dopamine hydrochloride and ammonium bicarbonate and incubating them together, and centrifuging and drying the obtained precipitate to obtain the hollow mineralized nano-calcium carbonate.

[0013] Further, the concentration of the dopamine hydrochloride is 0.01 - 0.1 mg / mL, and the incubation time is 12 - 48 h.

[0014] Preferably, the concentration of the dopamine hydrochloride is 0.03 - 0.07 mg / mL.

[0015] Further, the concentration of the calcium chloride dihydrate is 0.001 - 0.01 g / mL.

[0016] Preferably, the concentration of the calcium chloride dihydrate is 0.002 - 0.006 g / mL.

[0017] Further, before the hollow mineralized nano-calcium carbonate powder is mixed with γ-cyclodextrin, it is first filtered with a 0.45 filter membrane.

[0018] Furthermore, the potassium ion compound is potassium hydroxide. The reaction temperature between the cyclodextrin organic ligand and the potassium ion inorganic coordination ion is 35 - 50 °C, and the reaction time is 12 - 48 h. The reaction process is carried out in a methanol solution. When γ-cyclodextrin and calcium carbonate form MOF, at a relatively low temperature, the diffusion rate of methanol will be reduced, the yield of MOF will be decreased, and a large amount of calcium carbonate will settle and agglomerate, making it impossible to form MOF. At a relatively high temperature, the diffusion rate of methanol is too fast, resulting in difficulty in forming crystal nuclei. A large number of existing crystal nuclei will self-polymerize, making it difficult to form crystals. Moreover, high temperature will damage the internal structure of the crystal, resulting in damage to the hollow mineralized calcium carbonate structure and a conformational change of polydopamine.

[0019] Furthermore, the preparation process of the cyclodextrin MOF embedded in calcium carbonate is as follows: The hollow mineralized nano-calcium carbonate is uniformly dispersed in water. After ultrasonic dispersion, γ-cyclodextrin and the potassium ion compound are added. After ultrasonic treatment for 5 - 20 min, it is co-incubated with methanol for 12 - 72 h, and after centrifugation and drying, the cyclodextrin MOF embedded in calcium carbonate is obtained.

[0020] Furthermore, the mass ratio of the hollow mineralized nano-calcium carbonate powder to γ-cyclodextrin is 1:3 - 4. Preferably, the mass ratio of the hollow mineralized nano-calcium carbonate powder to γ-cyclodextrin is 10:31.6. In the process of forming MOF from γ-cyclodextrin and calcium carbonate, when the input amount of calcium carbonate is relatively low, the yield of MOF is low, and the morphology is unstable. Regarding the content of calcium carbonate in a single MOF, it is not sensitive to the change of pH. When the input amount of calcium carbonate is relatively high, there are more free calcium carbonates in the reaction solution, which are difficult to remove. The amount of calcium carbonate loaded in a single MOF is more, and the content of cyclodextrin is less, reducing the pore structure provided by cyclodextrin, which is not conducive to the subsequent loading and accommodation of drugs.

[0021] Furthermore, when grafting the RGD polypeptide, NHS, EDC, RGD and PBS are mixed and then added to the cyclodextrin MOF embedded in calcium carbonate, and the reaction is carried out at 4 °C for 1 - 8 h. After washing and drying, MOF-RGD is obtained.

[0022] Furthermore, the ratio of the thrombolytic drug added to MOF-RGD is 0.5 U:1 g - 100 U:1 g.

[0023] Furthermore, the thrombolytic drug is urokinase, lumbrokinase, streptokinase, nattokinase or tissue plasminogen activator; the anticoagulant drug is aspirin, dipyridamole, ticlopidine, clopidogrel, cilostazol, ozagrel, sarpogrelate, indobufen, prostacyclin, iloprost, dazoxiben, clofibrate, dextran 70, troxerutin, eptifibatide, tirofiban, abciximab, anagrelide or sulfinpyrazone.

[0024] The second object of the present invention is to provide an application of a nano-MOF with pH-responsive function for targeted dual-drug thrombolysis in the preparation of drugs with pH-responsive characteristics.

[0025] Furthermore, an application of a nano-MOF with pH-responsive function for targeted dual-drug thrombolysis in the preparation of drugs for the treatment and prevention of thrombosis is provided.

[0026] Furthermore, the thrombosis is venous thrombosis.

[0027] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0028] In the present invention, hollow mineralized nano-calcium carbonate is used as the substrate for responding to slightly acidic pH, and cyclodextrin is coated on the outer layer of nano-calcium carbonate to form a nano-cyclodextrin MOF as a shell structure, possessing various properties in the MOF. When entering the thrombus environment with slightly acidic pH, the calcium carbonate in the nano-MOF is broken, resulting in the "collapse" of the MOF structure, promoting the release of various functional raw materials loaded by the MOF, playing a role in thrombolysis and anticoagulation.

[0029] In view of the necessity of simultaneous fibrinolytic and anti-inflammatory treatment in the treatment of venous thrombosis in the present invention, special drugs such as aspirin are loaded in the cyclodextrin MOF skeleton, fibrinolytic drugs such as urokinase (UK) are loaded on the surface of the MOF, and cyclic RGD polypeptide is coupled at the same time to precisely target activated platelets, realizing the dual effects of first thrombolysis and then release of anti-inflammatory drugs at the thrombus. After entering the thrombus location, the MOF first releases thrombolytic drugs at the thrombus for thrombolysis, and the large release of thrombolytic drugs induces thrombolysis to restore vascular recanalization; the slightly acidic pH at the thrombus causes the calcium carbonate to break, and the material decomposes and releases aspirin, which reduces the aggregation of inflammatory factors at the thrombus and prevents re-embolism after thrombolysis.

[0030] The nano-MOF of the present invention uses cyclodextrin as the main carrier for drug loading and delivery, and hollow mineralized nano-calcium carbonate as the matrix for responding to slightly acidic pH. It is a porous pH-responsive functionalized porous MOF with a high specific surface area and good biocompatibility. The thrombolytic drugs on the MOF reach the slightly acidic environment of the thrombus under the action of RGD, and the slightly acidic environment causes the MOF to decompose, triggering the release of anticoagulant drugs, precisely targeting the thrombus site, and playing a dual role in thrombolysis and anticoagulation.

[0031] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly, it can be implemented according to the content of the specification. And in order to make the above and other objects, features and advantages of the present invention more obvious and understandable, the specific embodiments of the present invention are specifically given below. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1Flow chart of the preparation of a nano-MOF with pH-responsive function for targeted dual-drug thrombolysis and its preparation method according to the present invention.

[0033] Figure 2 Transmission electron microscope image and scanning electron microscope image of a nano-MOF with pH-responsive function for targeted dual-drug thrombolysis and its preparation method according to the present invention.

[0034] Figure 3 Particle size analysis chart of a nano-MOF with pH-responsive function for targeted dual-drug thrombolysis and its preparation method according to the present invention.

[0035] Figure 4 Effect diagram of the influence of pH on the release of a nano-MOF with pH-responsive function for targeted dual-drug thrombolysis and its preparation method according to the present invention.

[0036] Figure 5 Experimental diagram of thrombolysis effect of a nano-MOF with pH-responsive function for targeted dual-drug thrombolysis and its preparation method according to the present invention.

[0037] Figure 6 Experimental diagram of anticoagulant effect of a nano-MOF with pH-responsive function for targeted dual-drug thrombolysis and its preparation method according to the present invention.

[0038] Figure 7 Principle diagram of a nano-MOF with pH-responsive function for targeted dual-drug thrombolysis and its preparation method according to the present invention. Detailed implementation method

[0039] The technical solution of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following embodiments are only used to illustrate and explain the present invention exemplarily, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection of the present invention.

[0040] It should be noted that in the present invention, MOF refers to metal-organic framework; NHS is N-hydroxysuccinimide; EDC is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

[0041] In addition, unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present invention can be obtained through market purchase or prepared by existing methods.

[0042] Example 1:

[0043] See attached Figure 1 , a preparation method of a nano-MOF with pH-responsive function for targeted dual-drug thrombolysis, comprising the following steps:

[0044] 1) Preparation of hollow mineralized nano-calcium carbonate:

[0045] The hollow mineralized nano-calcium carbonate was prepared by a one-pot method.

[0046] Take 0.1 g of calcium chloride dihydrate and dissolve it in 100 mL of absolute ethanol. Then add 1 mg of dopamine hydrochloride and 5 g of ammonium bicarbonate and incubate them together in a sealed environment at 37 °C for 12 - 18 h to obtain a blue-black precipitate. Centrifuge at 8000 r for 10 min and dry it in an oven at 75 °C to obtain the hollow mineralized nano-calcium carbonate powder.

[0047] 2) Assembly of cyclodextrin MOF framework and hollow mineralized nano-calcium carbonate

[0048] The nano-cyclodextrin MOF embedded with calcium carbonate was prepared by the gas diffusion method. Uniformly disperse 40 mg of the nano-calcium carbonate powder prepared in step 1) in 10 mL of aqueous solution, ultrasonically disperse it, filter it with a 0.45 μm filter membrane, retain the filtrate, add 150 mg of γ-cyclodextrin and 60 mg of potassium hydroxide, ultrasonically disperse for 3 min, filter it with a filter membrane and co-incubate it with 20 mL of methanol. Slowly diffuse and react methanol in a sealed space for 12 - 24 h. After the reaction, ultrasonically disperse for 3 - 5 min and let it stand for layering. Use the gradient centrifugation method to separate the mixed phases with different particle sizes, wash the reactants three times with ethanol and acetone respectively, filter with a 0.45 μm filter membrane, centrifuge the product, and dry it at 45 °C for 3 h to obtain the pure cyclodextrin MOF embedded with calcium carbonate.

[0049] 3) Preparation of MOF-RGD

[0050] Mix NHS (1 mg / mL), EDC (1 mg / mL), RGD (1 mg / mL) and PBS evenly for 4 - 8 h, add the cyclodextrin MOF embedded with calcium carbonate prepared in step 2) (2 mg / mL) and react at 4 °C for 1 - 4 h. Wash it three times with PBS and then freeze-dry for 48 h to obtain the MOF grafted with RGD. After freeze-drying for 48 h, the product MOF-RGD is obtained.

[0051] 4) Impregnation of clopidogrel into MOF-RGD

[0052] Dissolve 10 mg of clopidogrel raw material in 3 mL of ethanol, impregnate 30 mg of the MOF-RGD obtained in step 3) into ethanol, shake it at room temperature for 8 h, wash it three times with absolute ethanol, and air-dry it at 37 °C to obtain the MOF-RGD particles loaded with clopidogrel.

[0053] Step 5) Loading of thrombolytic drugs

[0054] Mix streptokinase and MOF-RGD particles loaded with clopidogrel at a ratio of 10 U:1 g in PBS, shake at 4 °C for 30 min, repeat three times, and freeze-dry the precipitate at -50 °C for 48 h to obtain the pH-responsive targeted nano-MOF of the present invention.

[0055] To verify the effects of the concentration of dopamine hydrochloride and the feeding ratio of calcium carbonate to γ-cyclodextrin on the structure of nano-MOF, two groups of verification tests were carried out respectively.

[0056] In the first group, with other conditions unchanged, the amount of dopamine hydrochloride added was adjusted successively to 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, and 10 mg. It can be seen from the experimental results that when the amount of dopamine hydrochloride added was 1 mg - 3 mg, the particle size of the hollow mineralized nano-calcium carbonate powder was uneven. The more dopamine hydrochloride was added, the more uniform the particle size of the hollow mineralized nano-calcium carbonate powder was. When the amount of dopamine hydrochloride added reached 8 mg, the hollow mineralized nano-calcium carbonate agglomerated seriously, which was not conducive to the subsequent experiments. Therefore, the present invention determines that the optimal amount of dopamine hydrochloride added is 3 - 7 mg.

[0057] In the second group, with other conditions unchanged, only the amount of the hollow mineralized nano-calcium carbonate powder added was changed successively to 20 mg, 60 mg, 100 mg, 140 mg, and 180 mg, and the mass ratio of the nano-calcium carbonate powder to γ-cyclodextrin was limited to 1:3 - 4. It can be seen that the lower the amount of nano-calcium carbonate added, the more unstable the obtained MOF morphology was, and it was not sensitive to pH changes, which affected the drug effect. When the amount of nano-calcium carbonate added was relatively high, there was more free calcium carbonate, which was difficult to remove. More calcium carbonate was loaded on a single MOF, reducing the void structure of cyclodextrin, which was not conducive to the loading and accommodation of clopidogrel and streptokinase. Therefore, the present invention determines that the optimal amount of the hollow mineralized nano-calcium carbonate powder added is 60 - 140 mg, and the mass ratio of the hollow mineralized nano-calcium carbonate powder to γ-cyclodextrin is 10:31.6.

[0058] When γ-cyclodextrin and calcium carbonate form MOF, at a lower temperature, the diffusion rate of methanol will be reduced, the yield of MOF will be reduced, and a large amount of calcium carbonate will settle and agglomerate, resulting in the inability to form MOF. At a higher temperature, the diffusion rate of methanol is too fast, resulting in difficulty in forming crystal nuclei. A large number of existing crystal nuclei will self-polymerize, and it is difficult to form crystals. Moreover, high temperature will damage the internal structure of the crystal, resulting in damage to the structure of the hollow mineralized calcium carbonate and a change in the conformation of polydopamine. Control of the loading temperature of thrombolytic drugs. Thrombolytic drugs are easily inactivated at room temperature, and the loading temperature of thrombolytic drugs should be controlled at 0 - 4 °C to ensure that thrombolytic drugs are not affected by temperature.

[0059] Example 2:

[0060] A preparation method of a nano-MOF with pH-responsive function for targeted dual-drug delivery thrombolysis, comprising the following steps:

[0061] 1) Preparation of hollow mineralized nano-calcium carbonate:

[0062] Prepare hollow mineralized nano-calcium carbonate by a one-pot method

[0063] Take 0.2 g of calcium chloride dihydrate and dissolve it in 100 mL of absolute ethanol. Then add 4 mg of dopamine hydrochloride and 5 g of ammonium bicarbonate, and incubate them together in a sealed environment at 37 °C for 24 - 48 h to obtain a blue-black precipitate. Centrifuge at 8000 r for 10 min, and dry it in an oven at 75 °C to obtain hollow mineralized nano-calcium carbonate powder.

[0064] 2) Assembly of cyclodextrin MOF framework and hollow mineralized nano-calcium carbonate

[0065] Prepare nano-cyclodextrin MOF embedded with calcium carbonate by gas-phase diffusion method. Uniformly disperse 90 mg of the nano-calcium carbonate powder prepared in step 1) in 10 mL of aqueous solution, ultrasonically disperse it, filter it with a 0.45 filter membrane, retain the filtrate, add 300 mg of γ-cyclodextrin and 150 mg of potassium hydroxide, ultrasonically disperse for 5 - 8 min, filter it with a filter membrane and co-incubate with 20 mL of methanol. Slowly diffuse and react methanol in a sealed space for 24 - 48 h. After the reaction, ultrasonically disperse for 5 min and let it stand for layering. Use the gradient centrifugation method to separate the mixed phases with different particle sizes. Wash the reactants three times with ethanol and acetone respectively, filter with a 0.45-micron filter membrane, centrifuge the product, and dry it at 45 °C for 3 h to obtain pure cyclodextrin MOF embedded with calcium carbonate.

[0066] 3) Preparation of MOF-RGD

[0067] Mix NHS (2 mg / mL), EDC (1 mg / mL), RGD (2 mg / mL) and PBS evenly for 4 h, add 2 mg / mL of the cyclodextrin MOF embedded with calcium carbonate prepared in step 2), and react at 4 °C for 4 - 8 h. Wash it three times with PBS and then freeze-dry for 48 h to obtain MOF grafted with RGD. After freeze-drying for 48 h, obtain the product MOF-RGD.

[0068] 4) Impregnate aspirin into MOF-RGD

[0069] Dissolve 10 mg of aspirin raw material in 3 mL of ethanol. Immerse 40 - 60 mg of the MOF-RGD obtained in step 3) into ethanol, shake it at room temperature for 8 h, wash it three times with absolute ethanol, and air-dry it at 37 °C to obtain MOF-RGD particles loaded with aspirin.

[0070] Step 5) Loading of thrombolytic drugs

[0071] Urokinase was mixed with MOF-RGD particles loaded with aspirin at a ratio of 90 U:1 g in PBS, shaken at 4 °C for 30 min, and repeated three times. The precipitate was freeze-dried at -50 °C for 48 h to obtain the pH-responsive targeted nano-MOF of the present invention.

[0072] Experimental examples:

[0073] See the appendix Figure 2 , the pH-responsive targeted dual-drug delivery thrombolytic nano-MOF prepared in Example 2 was observed by transmission electron microscopy and scanning electron microscopy respectively. It can be seen from the transmission electron microscopy that the nano-MOF has a very obvious core-shell structure, and the hollow mineralized nano-calcium carbonate is embedded in the cyclodextrin MOF. It can be seen from the scanning electron microscopy that the nano-MOF is a cube structure with a uniform particle size distribution. From Figure 3 the particle size analysis chart, it can also be seen that the particle size of the nano-MOF is basically all concentrated between 100 and 300 nm, and the particles with a particle size of 200 to 300 nm account for 78%. It can be seen that the nano-calcium carbonate is successfully embedded in the MOF.

[0074] See the appendix Figure 4 , the disintegration of the pH-responsive targeted dual-drug delivery thrombolytic nano-MOF prepared in Example 2 was observed by transmission electron microscopy in various slightly acidic pH environments. From Figure 4 it can be seen that in the pH 7.4 environment, the nano-MOF did not disintegrate. In the pH 6.8 environment, the nano-MOF disintegrated, but the disintegration rate was slow; in the pH 6.5 environment, the nano-MOF disintegrated, and the disintegration rate was fast. It can be seen that in the normal venous blood pH environment of 7.35 - 7.45, the nano-MOF does not disintegrate, enabling the nano-MOF to be safely transported to the thrombus under the targeting action of the RGD polypeptide. Due to the slightly acidic pH environment, the nano-MOF disintegrates, releasing the drug to achieve the thrombolytic and anticoagulant effects.

[0075] See the appendix Figure 5, using fibrin plate characterization means, the thrombolysis situation was tested. Fibrin and agarose plates were prepared, and after standing at room temperature for 30 min until solidification, PBS, PBS containing urokinase, PBS containing the nano-MOF prepared in Example 2 of the present invention with a pH of 6.8, and PBS containing the nano-MOF prepared in Example 2 of the present invention with a pH of 7.4 were added into each vessel respectively, and the fibrin dissolution degree in the plate was observed. It can be seen that the PBS containing urokinase effectively dissolves thrombus; the PBS containing the nano-MOF prepared in Example 2 of the present invention with a pH of 6.8 effectively dissolves fibrin, and the nano-MOF is released; the PBS containing the nano-MOF prepared in Example 2 of the present invention with a pH of 7.4 also effectively dissolves fibrin, but the nano-MOF is not released. It can be seen that the urokinase in the nano-MOF has a thrombolytic effect before the calcium carbonate collapses, and the urokinase is located on the surface of the nano-MOF.

[0076] See Appendix Figure 6 , an in vitro thrombolysis experiment was carried out on the nano-MOF. Four groups of solutions were prepared, namely normal saline, normal saline and thrombin, normal saline, thrombin and nano-MOF prepared in Example 2 of the present invention with a pH of 7.4, and normal saline, thrombin and nano-MOF prepared in Example 2 of the present invention with a pH of 6.8. One drop of mouse whole blood was taken respectively, and after standing at room temperature for 30 min until coagulation, it was added into the four groups of solutions for observation. It can be seen that coagulation occurred in those without adding the nano-MOF of the present invention, and there was no hemolysis phenomenon; after adding the nano-MOF of the present invention, thrombolysis began after 1 hour at pH 7.4, and the thrombolysis phenomenon was obvious after 6 hours, and there was no re-coagulation phenomenon; after adding the nano-MOF of the present invention at pH = 6.8, an obvious thrombolysis phenomenon appeared after 1 hour, and after thrombolysis, the blood quickly spread and dispersed in normal saline, and was basically completely dissolved after 3 hours, with no obvious coagulation, and complete dissolution was achieved at 6 hours. It can be seen that the aspirin released in a hierarchical manner by the nano-MOF of the present invention has a good anticoagulant effect and a good anticoagulant effect after thrombolysis.

[0077] Appendix Figure 7This is the schematic diagram of the thrombolytic and anticoagulant effects of the nano-MOF of the present invention. Combining the above experiments, it can be seen that the present invention has a pH-responsive function of targeted dual-drug delivery thrombolytic nano-MOF. Using the crystal growth principle of MOF, with hollow mineralized nano-calcium carbonate as the responsive substrate and cyclodextrin MOF as the shell structure, a drug-loading structure of cyclodextrin-coated hollow porous calcium carbonate nano-MOF is constructed. The nano-MOF of the present invention has various properties of MOF. When the nano-MOF enters the vein in the form of intravenous injection, etc., under the precise targeting effect of the RGD polypeptide, it moves towards the thrombus site. When it enters the slightly acidic pH thrombus environment, the urokinase on the surface of the nano-MOF is first released to thrombolyze the thrombus. After the urokinase is released, the calcium carbonate in the nano-MOF will cause the nano-MOF structure to collapse in the slightly acidic environment, and the drugs such as aspirin carried by the nano-MOF are released. Then the aspirin inside the cyclodextrin MOF is released, and the massive release of urokinase induces thrombolysis to restore vascular recanalization. Aspirin reduces the aggregation of inflammatory factors at the thrombus, preventing re-embolism after thrombolysis, thus realizing the dual effects of thrombolysis first and then release of anticoagulant drugs at the thrombus site.

[0078] It should be noted that the thrombolytic drugs in the present invention are not limited to urokinase, lumbrokinase, streptokinase, nattokinase, tissue plasminogen activator, etc., and the anticoagulant drugs are not limited to aspirin, dipyridamole, ticlopidine, clopidogrel, cilostazol, ozagrel, sarpogrelate, indobufen, prostacyclin, iloprost, dazoxiben, clofibrate, dextran 70, troxerutin, eptifibatide, tirofiban, abciximab, anagrelide, sulfinpyrazone, etc. All kinds of thrombolytic drugs with thrombolytic effects and all kinds of drugs with anticoagulant effects are within the protection scope of the present invention.

[0079] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0080] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are only illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the purpose of the present invention and the scope protected by the claims, and these all belong to the protection scope of the present invention.

Claims

1. A nano-MOF with pH-responsive function for targeted dual-drug delivery thrombolysis, characterized in that: It includes cyclodextrin MOF, hollow mineralized nano-calcium carbonate embedded in the cyclodextrin MOF, RGD with targeting effect, anticoagulant drugs loaded in the cavities and pores of the cyclodextrin MOF, and thrombolytic drugs loaded on the surface of the MOF.

2. A preparation method of a nano-MOF with pH-responsive function for targeted dual-drug thrombolysis according to claim 1, characterized in that: The hollow mineralized nano-calcium carbonate powder is mixed with γ-cyclodextrin and potassium ion compound, ultrasonically dispersed and then incubated to form cyclodextrin MOF embedded with calcium carbonate; RGD polypeptide is grafted on the surface of the cyclodextrin MOF embedded with calcium carbonate to obtain MOF-RGD; the grafted MOF-RGD is impregnated into ethanol dissolved with anticoagulant drugs, air-dried and then mixed with thrombolytic drugs, so that the thrombolytic drugs are loaded on the surface of the nano-MOF to obtain pH-responsive targeted nano-MOF.

3. The preparation method of a nano-MOF for targeted dual-drug delivery thrombolysis with pH-responsive function as claimed in claim 2, wherein, The preparation method of the hollow mineralized nano-calcium carbonate is as follows: calcium chloride dihydrate is dissolved in absolute ethanol, dopamine hydrochloride and ammonium bicarbonate are added and incubated together, and the obtained precipitate is centrifuged and dried to obtain the hollow mineralized nano-calcium carbonate.

4. The preparation method of a nano-MOF with pH-responsive function for targeted dual-drug thrombolysis according to claim 2, characterized in that, The preparation process of the cyclodextrin MOF embedded with calcium carbonate is as follows: the hollow mineralized nano-calcium carbonate is uniformly dispersed in water, ultrasonically dispersed, then γ-cyclodextrin and potassium ion compound are added, ultrasonically treated for 3 min and then co-incubated with methanol for 12 - 48 h, and after centrifugation and drying, the cyclodextrin MOF embedded with calcium carbonate is obtained.

5. The preparation method of a nano-MOF with pH-responsive function for targeted dual-drug thrombolysis as described in claim 2, characterized in that: The mass ratio of the hollow mineralized nano-calcium carbonate powder to γ-cyclodextrin is 1:3 - 4.

6. The preparation method of a nano-MOF with pH-responsive function for targeted dual-drug thrombolysis as described in claim 2, characterized in that: When grafting RGD polypeptide, NHS, EDC, RGD and PBS are mixed and then added to the cyclodextrin MOF embedded with calcium carbonate, and the reaction is carried out at 4 °C for 8 h, and after washing and drying, MOF-RGD is obtained.

7. The preparation method of a nano-MOF with pH-responsive function for targeted dual-drug thrombolysis according to claim 2, characterized in that: The ratio of the added thrombolytic drug to MOF-RGD is 0.5 U:1 g - 100 U:1 g.

8. Use of a pH-responsive targeted dual-drug delivery thrombolytic nano-MOF prepared by the method according to any one of claims 2 - 7 in the preparation of drugs for treating and preventing thrombosis.

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