A binuclear iridium metal conjugate, water-soluble nanoparticles, preparation method and application in ultrasonic thrombolysis

Abstract

This invention provides a binuclear iridium metal conjugate, nanoparticles, a preparation method, and its application in ultrasonic thrombolysis, belonging to the field of sonodynamic therapeutic drug technology. The structural formula of the binuclear iridium metal conjugate is shown in formula (I). This invention also provides a method for preparing the above-mentioned binuclear iridium metal conjugate. This invention also provides nanoparticles. The binuclear iridium metal conjugate prepared by this invention possesses chemiluminescent properties, enabling chemiluminescence imaging-assisted ultrasound-assisted thrombolysis. The conjugate provided by this invention exhibits excellent sonodynamic effects, overcoming the poor sonodynamic performance required by existing sonosensitive agents. Experiments show that this material has excellent therapeutic effects in thrombolysis.

Description

Technical Field

[0001] This invention belongs to the field of sonodynamic therapeutic drug technology, specifically relating to a binuclear iridium metal conjugate, nanoparticles, preparation method, and application in ultrasonic thrombolysis. Background Technology

[0002] Thrombosis occurs when a blood vessel is partially or completely blocked by a clot, potentially leading to acute ischemic stroke, coronary infarction, or pulmonary embolism, a leading cause of death and long-term disability worldwide. Pharmacological thrombolysis, involving antiplatelet or fibrinolytic drugs, brings a variety of side effects, including potential neurotoxicity and fatal bleeding complications, with increased bleeding being a leading cause of death. Therefore, there is an urgent need to develop a comprehensive thrombosis treatment approach to achieve precision medicine.

[0003] Sonodynamic therapy (SDT) is considered a promising approach due to its deep tissue penetration and the site-specific (ultrasound) characteristics of ultrasound. SDT refers to the generation of singlet oxygen via ultrasound-activated sonosensitive agents to achieve thrombolysis without the combined action of tPA. Reactive oxygen species (ROS) can disrupt the phospholipid and fibrin backbone, thereby dissolving thrombi. The enhanced spin-orbit coupling promoted by the heavy atom effect in the sonosensitive agent can also increase the intersystem crossover rate, thereby promoting the generation of free radicals. Ir(III) complexes have attracted much attention due to their desirable photophysical properties, large Stokes shift, and high ISC capacity. Furthermore, the emission properties of iridium(III) complexes, especially heterocyclic metallized Ir(III) complexes, can be effectively tuned through ligand design.

[0004] Currently, the application of afterglow luminescent probes based on single-molecule Ir(III) complexes has not been fully validated, and there are few reports on drug design for chemiluminescence imaging-guided therapy against thrombi. This work demonstrates the great potential for precise diagnosis and treatment of minimal lesions, and has significant implications for clinical selection of thrombus therapy. Summary of the Invention

[0005] The purpose of this invention is to provide a binuclear iridium metal conjugate, water-soluble nanoparticles, a preparation method, and its application in ultrasonic thrombolysis. Compared with mononuclear iridium metal conjugates, the binuclear iridium metal conjugate of this invention has better reactive oxygen species generation capacity and chemiluminescence properties, and exhibits excellent therapeutic effects in in vitro and in vivo experiments.

[0006] This invention first provides a dual-core iridium metal coupling, the structural formula of which is shown in formula (Ⅰ): (I).

[0007] The present invention also provides a method for preparing the above-mentioned binuclear iridium metal conjugate, comprising the following steps: S1. Under nitrogen protection, IrCl3·3H2O and 2-phenylbenzimidazole ligand were heated under reflux to obtain 2-phenylbenzimidazole iridium dichlorobridge [Ir(DPIM)2Cl2]2; S2. A solution of nickel chloride and triphenylphosphine was placed in N,N-dimethylformamide and heated under a nitrogen atmosphere to obtain a blue suspension. Zinc powder was then added to form a reddish-brown suspension. 5-bromo-2,2'-bipyridine was then added and stirred to obtain 5-(5-bipyridine-2,2'-yl)-2,2'-bipyridine ligand. S3. Under nitrogen protection, the 2-phenylbenzimidazole iridium dichlorobridge prepared in S1 and the 5-(5-bipyridin-2,2'-yl)-2,2'-bipyridinium ligand prepared in S2 were placed in the dark and subjected to reflux reaction in the presence of a solvent. After the reaction was completed, potassium hexafluorophosphate was added and the mixture was stirred to obtain a binuclear iridium metal conjugate.

[0008] Preferably, in step S2, the molar ratio of nickel chloride, triphenylphosphine, zinc powder and 5-bromo-2,2'-bipyridine is 1.34:5.1:1.38:1.27.

[0009] Preferably, in step S2, the heating temperature under a nitrogen atmosphere is 50°C and the heating time is 0.5 h.

[0010] Preferably, in step S3, the molar ratio of the 2-phenylbenzimidazole iridium dichlorobridge and the 5-(5-bipyridin-2,2'-yl)-2,2'-bipyridinium ligand is 1:1.

[0011] Preferably, in step S3, the reflux reaction temperature is 65~80℃ and the reflux reaction time is 6 h.

[0012] The present invention also provides a nanoparticle prepared using the above-mentioned binuclear iridium metal conjugate.

[0013] The present invention also provides a method for producing nanoparticles, comprising: polyethylene glycol 2000 -MAL and arginine-glycine-aspartic peptide c (RGDfC) were dissolved in tetrahydrofuran and stirred. Then, a tetrahydrofuran solution of the binuclear iridium metal conjugate Ir-2 was added and mixed. The mixture was then added dropwise to ultrapure water and stirred. After dialysis, nanoparticles Ir-2@R NPs were obtained.

[0014] The present invention also provides the application of the above-mentioned nanoparticles as sound therapy materials.

[0015] The present invention also provides the application of the above-mentioned nanoparticles in the preparation of drugs for treating thrombotic diseases.

[0016] Beneficial effects of the present invention This invention provides a binuclear iridium metal conjugate, water-soluble nanoparticles, a preparation method, and its application in ultrasonic thrombolysis. The conjugate Ir-2 synthesized in this invention, by introducing the chemiluminescent active material structure 2-phenylbenzimidazole and a Schiff base ligand, successfully constructs a binuclear iridium metal conjugate with excellent reactive oxygen species (ROS) generation and chemiluminescent properties. Under chemiluminescent imaging guidance, Ir-2 can effectively dissolve thrombi through its excellent acoustic dynamic properties. This invention also provides nanoparticles Ir-2@R NPs formed from the binuclear iridium metal conjugate Ir-2. Ir-2@R NPs exhibit excellent acoustic dynamic properties and can effectively dissolve thrombi. Attached Figure Description

[0017] Figure 1 The mass spectrometry data of Ir-2 prepared in Example 1 of this invention; Figure 2 The particle size potential diagram of the Ir-2@R NPs prepared in this invention; Figure 3 The UV absorption spectrum of the Ir-2@R NPs prepared in this invention in aqueous solution; Figure 4 AIE performance test graph of Ir-2@R NPs prepared for this invention under the condition of tetrahydrofuran as a good solvent and water as a poor solvent; Figure 5 The DPBF decrease curve for the experiment of singlet oxygen generation in the aqueous solution of Ir-2@R NPs prepared in this invention. Figure 6 The graph shows the blood compatibility test data of the Ir-2@R NPs prepared in this invention. Figure 7 The image shows thrombolysis data of Ir-2@R NPs prepared in this invention under different conditions; Figure 8 This is a test image of Ir-2@R NPs mouse thrombosis imaging prepared according to the present invention; Figure 9 The thrombolysis data of Ir-2@R NPs prepared in this invention in mice under different conditions is shown in the figure. Figure 10 The images show H&E slice data of blood vessels and mouse organs of the Ir-2@R NPs prepared in this invention. Detailed Implementation

[0018] This invention first provides a bimetallic iridium metal coupling, the structural formula of which is shown in formula (Ⅰ): (I).

[0019] The present invention also provides a method for preparing the above-mentioned binuclear iridium metal conjugate, comprising the following steps: S1. IrCl3·3H2O and 2-phenylbenzimidazole ligand are added to a reaction vessel containing solvent and water. The reaction is carried out under nitrogen protection and refluxed, preferably at 120~130℃ for 24~30 h. After the reaction is cooled to room temperature, a large amount of undesirable solvent water is added to precipitate the precipitate, and the precipitate is filtered. The solvent is washed away with a large amount of water and ethanol. The resulting solid is dried to obtain 2-phenylbenzimidazole iridium dichlorobridge [Ir(DPIM)2Cl2]2. The molar ratio of IrCl3·3H2O to 2-phenylbenzimidazole is preferably 1: (2.5~3), and the solvent is preferably 2-ethylene glycol ethyl ether. S2. Nickel chloride and triphenylphosphine are placed in N,N-dimethylformamide and heated at 50°C for 0.5 hours under a nitrogen atmosphere to obtain a blue suspension. Zinc powder is then added, and after 0.5 hours, a reddish-brown suspension forms. 5-Bromo-2,2'-bipyridine is then added, and the mixture is stirred at room temperature for 3 days. Next, 0.25 mol / L sodium ethylenediaminetetraacetate is added, the aqueous layer is washed with dichloromethane, and then saturated brine is added. The organic layers are combined, dried over magnesium sulfate, and the solvent is removed under reduced pressure. The product is then purified by rapid silica gel column chromatography. The resulting product is recrystallized in hot acetonitrile to obtain white plate-like crystals, yielding the 5-(5-bipyridine-2,2'-yl)-2,2'-bipyridine ligand. The preferred molar ratio of nickel chloride, triphenylphosphine, zinc powder, and 5-bromo-2,2'-bipyridine is 1.34:5.1:1.38:1.27.

[0020] S3. Add the obtained 2-phenylbenzimidazole iridium dichlorobridged [Ir(DPIM)2Cl2]2 and 5-(5-bipyridin-2,2'-yl)-2,2'-bipyridinium ligand to the reaction vessel, then add a solvent, preferably a mixture of dichloromethane and methanol (v:v=1:1). Under sufficient purging with inert gas N2, place the reaction vessel in the dark and reflux it. The reflux temperature is preferably 65-80°C, and the reaction time is preferably 6 h. After the reaction is complete and cooled to room temperature, add potassium hexafluorophosphate solid to the solution in the flask and continue stirring at room temperature. The stirring time is preferably 45-60 seconds. The solvent in the system was removed by rotary evaporation, followed by extraction with dichloromethane and water to remove excess potassium hexafluorophosphate solid. The obtained substance was washed with petroleum ether and dried, and purified by column chromatography to obtain a red solid, namely the binuclear iridium metal conjugate Ir-2. The molar ratio of the 2-phenylbenzimidazole iridium dichlorobridge [Ir(DPIM)2Cl2]2 and the 5-(5-bipyridin-2,2'-yl)-2,2'-bipyridine ligand is preferably 1:1; the molar ratio of the iridium metal complex Ir-2 and potassium hexafluorophosphate is preferably 1:5.

[0021] The synthetic route of this preparation method is shown below:

[0022]

[0023]

[0024] The present invention also provides a nanoparticle prepared using the above-mentioned binuclear iridium metal coupling compound, wherein the particle size of the nanoparticle is preferably 100~200 nm.

[0025] The present invention also provides a method for producing nanoparticles, comprising: polyethylene glycol 2000 -MAL and c(RGDfC) are dissolved in tetrahydrofuran and stirred for two hours. Then, a tetrahydrofuran solution of the binuclear iridium metal conjugate Ir-2 is added to the mixture, and the solution is added dropwise to ultrapure water. Stirring is preferably carried out at room temperature for 8 hours. Dialysis yields Ir-4@R NPs nanoparticles. The polyethylene glycol... 2000 The preferred mass ratio of MAL, c(RGDfC), and the binuclear iridium metal coupling compound Ir-2 is 1:0.5:0.5.

[0026] The present invention also provides the application of the above-mentioned nanoparticles as a sound therapy material, wherein the sound therapy material is preferably a sound-sensitive initiator.

[0027] The present invention also provides the application of the above-mentioned nanoparticles in ultrasonic thrombolytic drugs for diseases.

[0028] Unless otherwise stated, the terms used herein have the meanings conventionally understood by those skilled in the art. The technical solutions of the present invention will be described in more detail below with reference to embodiments, in which all raw materials are commercially available.

[0029] Example 1 S1. IrCl3·3H2O (0.1 mmol, 0.0352 g) and 2-phenylbenzimidazole ligand (0.3 mmol, 0.0582 g) were added to a round-bottom flask containing 30 mL of 2-ethylene glycol ethyl ether and 10 mL of water. The mixture was heated to reflux at 120 °C for 24 h under a nitrogen-protected atmosphere. After the reaction was cooled to room temperature, a large amount of water, a poor solvent, was added to precipitate the solid. The precipitate was then filtered and washed repeatedly with large amounts of water and ethanol. The resulting solid, when dried, was 2-phenylbenzimidazole iridium dichlorobridge [Ir(DPIM)2Cl2]2.

[0030] S2. Nickel chloride (0.174 g, 1.34 mmol) and triphenylphosphine (1.34 g, 5.10 mmol) were placed in 12 mL of N,N-dimethylformamide and heated at 50 °C for 0.5 h under a nitrogen atmosphere to obtain a blue suspension. Zinc powder (90.3 mg, 1.38 mmol) was then added, and after 0.5 h, a reddish-brown suspension formed. 5-bromo-2,2'-bipyridine (0.299 g, 1.27 mmol) was then added, and the mixture was stirred at room temperature for 3 days. Next, 0.25 mol / L sodium ethylenediaminetetraacetate was added, and the aqueous layer was washed with dichloromethane followed by the addition of saturated brine. The organic layers were combined, dried over magnesium sulfate, and the solvent was removed under reduced pressure. The product was purified by rapid silica gel column chromatography. The resulting product was recrystallized in hot acetonitrile to obtain white plate-like crystals, yielding the 5-(5-bipyridine-2,2'-yl)-2,2'-bipyridine ligand.

[0031] S3. Add the obtained 2-phenylbenzimidazole iridium dichlorobridged [Ir(DPIM)2Cl2]2 (0.1 mmol, 0.1832 g) and 5-(5-bipyridin-2,2'-yl)-2,2'-bipyridine (0.1 mmol, 0.031 g) to a 100 mL single-necked flask, then add 60 mL of dichloromethane and methanol (v:v=1:1) as solvents for the reaction system. Under the condition of fully purging with inert gas N2, place the reaction in the dark and reflux for 6 h at 80 °C. After the reaction is completed and cooled to room temperature, add 200 mg of potassium hexafluorophosphate solid to the solution in the flask, continue stirring at room temperature for 45 min, remove the solvent from the system using a rotary evaporator, extract with dichloromethane and water to remove excess potassium hexafluorophosphate solid, wash the obtained substance with petroleum ether and dry to obtain a red solid, namely Ir-2. The yield was 84%, and the molecular formula was C. 72 H 50 Ir2N 12 Its relative molecular mass is 1468.35 g / mol. Figure 1 The mass spectrometry data for the prepared Ir-2 shows a molecular weight of 1468.36 g / mol, which is the same as the fitted molecular weight, proving that its structure was successfully prepared.

[0032] Example 2 The experimental conditions and steps were the same as in Example 1, except that in step S1, the reaction was stirred at 125°C for 24 hours.

[0033] Example 3 The experimental conditions and steps were the same as in Example 1, except that in step S3, the reaction was stirred at 75°C for 6 hours.

[0034] The performance of the iridium metal conjugate Ir-2 prepared in Example 1 was characterized as follows: 1. Preparation of nanoparticles 2g of polyethylene glycol 2000 -MAL and 1g c(RGDfC) were dissolved in tetrahydrofuran and stirred for two hours. Then, a tetrahydrofuran solution of 1g iridium metal coupling compound Ir-2 was added to the mixture and added dropwise to 10mL of ultrapure water. The mixture was stirred at room temperature for 8 hours and then dialyzed to obtain nanoparticles Ir-2@R NPs.

[0035] 2. Physical properties of Ir-2@R NPs Figure 1 The mass spectrometry data of Ir-2 is shown, which matches the theoretical values ​​to prove the structure. Figure 2The image shows the particle size of Ir-2@R NPs (measured by a particle size potentiometer). The smaller image represents a transmission electron microscope (TEM) image of Ir-2@R NPs. As can be seen from the image, the particle size of Ir-2@R NPs in aqueous solution is 146.2 nm.

[0036] 3. Photophysical properties of Ir-2@R NPs The UV absorption spectrum of Ir-2@RNPs in aqueous solution was measured using a UV-Vis spectrophotometer, and the results are as follows: Figure 3 As shown. Among them. Figure 3 This is a UV absorption spectrum. Figure 3 This indicates that the maximum ultraviolet absorption of Ir-2 can reach around 500 nm.

[0037] Figure 4 The emission of Ir-2@RNPs prepared for this invention in dimethyl sulfoxide. Figure 4 This indicates that the fluorescence emission peak of Ir-2@R NPs is around 670 nm.

[0038] Figure 5 The Ir-2@R NPs prepared in this invention were subjected to in vitro singlet oxygen generation experiments and related kinetic curves under illumination and DPBF conditions measured by ultraviolet spectrophotometer. The DPBF decrease line graph at a specific nanometer and the degradation of DPBF under ultrasonication prove that Ir-2@R NPs have good singlet oxygen generation ability.

[0039] 4. Blood compatibility test of Ir-2@R NPs Figure 6 This figure shows the blood compatibility test data of the Ir-2@R NPs prepared in this invention. Different concentrations of Ir-2@RNPs were incubated with blood cells for 2 hours, and the supernatant was collected for testing to calculate the hemolysis rate. As shown in the figure, the hemolysis rate of Ir-2@RNPs was less than 5%, indicating that Ir-2@R NPs have good blood compatibility and can be used for in vivo testing.

[0040] 5. In vitro thrombolysis experiment of Ir-2@R NPs 10 mL of different treatment groups and thrombus blocks were placed together in 20 mL vials for in vitro thrombolysis testing. The results are as follows: Figure 7 As shown. Figure 7 The thrombolysis data of Ir-2@R NPs prepared in this invention under different conditions are shown in the figure. (The preparation method of Ir-1@R NPs is the same as that of Ir-2@R NPs, except that Ir-2 is replaced by Ir-1, and the structural formula of Ir-1 is...) ).in Figure 7 (a) and Figure 7(b) A graph showing the changes in thrombus size and solution color before and after thrombolysis, in which... Figure 7 (c) shows the thrombus dissolution rate. Compared with the control group, the thrombus dissolution rate of the Ir-2@R NPs group reached 74.3% under ultrasound. The experimental results indicate that Ir-2@R NPs has good in vitro thrombolytic ability. Figure 7 (d) shows the fibrin and hemoglobin levels measured in the supernatant after thrombolysis, which show the same trend as the thrombolysis results in 7 (b).

[0041] 6. In vivo mouse experiments with Ir-2@R NPs Figure 8 This is a test image of Ir-2@R NPs mouse thrombosis imaging monitoring prepared according to the present invention. Figure 8 (a) is a graph of chemiluminescence signals detected by a small animal imaging system. Figure 8 (b) is a quantitative analysis diagram of chemiluminescence intensity of 8 (a). It can be seen that after administration via the tail vein, as time goes on, the drug circulates to the thrombus site, and the chemiluminescence phenomenon is obvious. The aggregation reaches its maximum at 60 min, and thrombolytic therapy can be started.

[0042] Figure 9 This image shows thrombolytic data of Ir-2@R NPs prepared in this invention under different conditions in mice. Mice were anesthetized and the right carotid artery was located via thoracotomy. The carotid artery was incubated with 10% FeCl3 solution for approximately 5 minutes, causing the vessel to turn black, indicating thrombolysis was complete. After drug administration, the drug circulated to the thrombus site, and ultrasound (1.0 W cm⁻¹) was used to measure the thrombus. -2 Irradiating the thrombus site with 1 MHz (1 MHz) has a good thrombolytic effect.

[0043] Figure 10 The images show H&E slice data of blood vessels and mouse organs of the Ir-2@R NPs prepared in this invention. Figure 10 This is a H&E section of blood vessels and mouse heart, liver, spleen, lungs and kidneys after thrombolysis. Figure 10 (a) The vascular section clearly shows that after thrombolysis, the plaque in the blood vessel is significantly reduced, proving the good thrombolytic effect of Ir-2@R NPs. Figure 10 (b) The mouse organ sections were all normal, demonstrating that Ir-2@R NPs have good biocompatibility.

[0044] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A binuclear iridium metal coupling, characterized in that, The structural formula of the metal coupling is shown in formula (Ⅰ): （Ⅰ）。 2. The method for preparing the above-mentioned binuclear iridium metal coupling according to claim 1, characterized in that, Includes the following steps: S1. Under nitrogen protection, IrCl3·3H2O and 2-phenylbenzimidazole ligand were heated under reflux to obtain 2-phenylbenzimidazole iridium dichlorobridge [Ir(DPIM)2Cl2]2; S2. A solution of nickel chloride and triphenylphosphine was placed in N,N-dimethylformamide and heated under a nitrogen atmosphere to obtain a blue suspension. Zinc powder was then added to form a reddish-brown suspension. 5-bromo-2,2'-bipyridine was then added and stirred to obtain 5-(5-bipyridine-2,2'-yl)-2,2'-bipyridine ligand. S3. Under nitrogen protection, the 2-phenylbenzimidazole iridium dichlorobridge prepared in S1 and the 5-(5-bipyridin-2,2'-yl)-2,2'-bipyridinium ligand prepared in S2 were placed in the dark and subjected to reflux reaction in the presence of a solvent. After the reaction was completed, potassium hexafluorophosphate was added and the mixture was stirred to obtain a binuclear iridium metal conjugate.

3. The method for preparing the above-mentioned binuclear iridium metal coupling compound according to claim 2, characterized in that, In step S2, the molar ratio of nickel chloride, triphenylphosphine, zinc powder and 5-bromo-2,2'-bipyridine is 1.34:5.1:1.38:1.

27.

4. The method for preparing the above-mentioned binuclear iridium metal coupling compound according to claim 2, characterized in that, In step S2, the heating temperature under a nitrogen atmosphere is 50°C and the heating time is 0.5 h.

5. A method for preparing the above-mentioned binuclear iridium metal coupling according to claim 2, characterized in that, In step S3, the molar ratio of the 2-phenylbenzimidazole iridium dichlorobridge and the 5-(5-bipyridin-2,2'-yl)-2,2'-bipyridinium ligand is 1:

1.

6. The method for preparing the above-mentioned binuclear iridium metal coupling according to claim 2, characterized in that, In step S3, the reflux reaction temperature is 65~80℃, and the reflux reaction time is 6 h.

7. A nanoparticle, characterized in that, The nanoparticles were prepared using the binuclear iridium metal conjugate described in claim 1.

8. The method for preparing nanoparticles according to claim 7, characterized in that, include: polyethylene glycol 2000 -MAL and arginine-glycine-aspartic peptide c (RGDfC) were dissolved in tetrahydrofuran and stirred. Then, a tetrahydrofuran solution of the binuclear iridium metal conjugate Ir-2 was added and mixed. The mixture was then added dropwise to ultrapure water and stirred. After dialysis, nanoparticles Ir-2@R NPs were obtained.

9. The application of the nanoparticles as a sound therapy material according to claim 7.

10. The use of the nanoparticles according to claim 7 in the preparation of drugs for treating thrombotic diseases.

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