Iridium metal conjugate, water-soluble nanoparticle, preparation method and application in ultrasonic thrombolysis

Abstract

The invention provides an iridium metal conjugate, a water-soluble nanoparticle, a preparation method and application in ultrasonic thrombolysis, and belongs to the technical field of sonodynamic therapeutic drugs. The structural formula of the iridium metal conjugate is as shown in formula (I). The invention also provides a preparation method of the iridium metal conjugate. The invention also provides a nanoparticle, and the iridium metal conjugate prepared by the invention has a chemiluminescence property, so that the iridium metal conjugate can realize chemiluminescence imaging-assisted ultrasonic synergistic thrombolysis therapy. The conjugate provided by the invention has a good sonodynamic effect, and can overcome the defect of poor sonodynamic effect required by the existing sonosensitizer. Experiments show that the material has a good treatment effect in thrombolysis treatment.

Description

Technical Field

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

[0002] Intravascular thrombosis (blood clots) is a major contributing factor to numerous serious cardiovascular diseases, which are the leading causes of disease and death worldwide. Current treatments for thrombosis primarily include drug therapy and surgery. However, both methods have limitations and drawbacks. Surgical treatment is highly effective but invasive and risky. Drug therapy has developed rapidly but often leads to bleeding complications, has a short half-life, and lacks specificity. Therefore, there is an urgent need to develop a comprehensive thrombosis treatment method to achieve precision medicine.

[0003] Sonodynamic therapy (SDT) is a promising treatment for thrombosis due to its non-invasive nature, deep tissue penetration, and precise targeting, based on the mechanism of ultrasound (US). However, localized ultrasound energy alone may not be effective in dissolving thrombi without the addition of thrombolytic drugs. Sonosensitive agents are activated by ultrasound, transitioning from their ground state to an excited state. These agents typically undergo intersystemic crossover (ISC) to enter a longer-lived triplet state, thus transforming the ground state triplet state... 3 O2 is converted into an excited singlet state. 1 O2. Alternatively, ultrasound-activated sonosensitive agents can directly interact with the biological matrix, gaining a hydrogen atom or an electron, thereby generating free radicals. Reactive oxygen species (ROS) can disrupt phospholipids and fibrin backbones, thus dissolving thrombi. The enhanced spin-orbit coupling promoted by the heavy atom effect in sonosensitive agents can also increase the intersystem crossover rate, thereby promoting free radical generation. The low water solubility of sonosensitive agents leads to reduced free radical generation due to aggregation-induced quenching (ACQ). However, the opposite phenomenon of aggregation-induced emission (AIE) can enhance fluorescence and reactive oxygen species (ROS) generation in the aggregated state by restricting intramolecular motion, thereby reducing energy loss. Iridium (III) complexes are a novel type of sonosensitive agent that has attracted much attention due to their excellent ROS generation ability, high acoustic stability, and tunable ligand modification.

[0004] Currently, there are still limited metal complexes suitable for integrated thrombolytic therapy, and there are few reports on drug design targeting the thrombus microenvironment. Providing more metal complexes that can be used for imaging-guided therapy is of great significance for clinical selection of thrombosis treatment. Summary of the Invention

[0005] The purpose of this invention is to provide an iridium metal conjugate, water-soluble nanoparticles, a preparation method, and its application in ultrasonic thrombolysis. The iridium metal conjugate of this invention has chemiluminescent properties, enabling visual monitoring in the early stages of disease development, and its excellent acoustic dynamic properties can effectively dissolve thrombi.

[0006] This invention first provides an 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 iridium metal coupling, comprising the following steps: S1. Under nitrogen protection, IrCl3·3H2O and quinoline triphenylamine ligand were heated under reflux to obtain quinoline triphenylamine iridium dichlorobridge [Ir(TPA)2Cl2]2; S2. Under nitrogen protection, the quinoline triphenylamine iridium dichlorobridge and 2-(2-pyridine)benzimidazole ligand prepared in S1 were placed in the dark and refluxed in the presence of a solvent. After the reaction was completed, potassium hexafluorophosphate was added and the mixture was stirred to obtain the iridium metal conjugate Ir-4.

[0008] Preferably, in step S2, the molar ratio of the quinoline triphenylamine iridium dichlorobridge and the 2-(2-pyridine)benzimidazole ligand is 1:2.

[0009] Preferably, in step S2, the reflux reaction temperature is 65~80℃ and the reflux reaction time is 6 h.

[0010] The present invention also provides a nanoparticle prepared using the above-mentioned iridium metal coupling compound.

[0011] The present invention also provides a method for producing nanoparticles, comprising: polyethylene glycol 2000 -MAL and c(RGDfC) were dissolved in tetrahydrofuran and stirred. Then, a tetrahydrofuran solution of iridium metal coupling compound Ir-4 was added and mixed. The mixture was then added dropwise to ultrapure water and stirred. After dialysis, nanoparticles Ir-4@R NPs were obtained.

[0012] Preferably, the stirring temperature is room temperature and the stirring time is 8 hours.

[0013] Preferably, the polyethylene glycol 2000 The mass ratio of MAL, c(RGDfC) and iridium metal coupling is 2:1:1.

[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 an iridium metal conjugate, water-soluble nanoparticles, a preparation method, and their application in ultrasonic thrombolysis. The conjugate Ir-4 synthesized in this invention couples a chemiluminescent active substance to an iridium complex, giving it chemiluminescent properties and excellent acoustic-dynamic performance. This invention also provides nanoparticles Ir-4@R NPs formed from the iridium metal conjugate Ir-4. Ir-4@R NPs exhibit excellent acoustic-dynamic properties and can effectively dissolve thrombi. Attached Figure Description

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

[0018] This invention first provides an 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 iridium metal coupling, comprising the following steps: S1. IrCl3·3H2O and quinoline triphenylamine ligand are added to a reaction vessel containing solvent and water. The reaction is carried out under nitrogen protection and heated under reflux, 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 quinoline triphenylamine iridium dichlorobridge [Ir(TPA)2Cl2]2. The molar ratio of IrCl3·3H2O to quinoline triphenylamine is preferably 1: (2.5~3), and the solvent is preferably 2-ethylene glycol ethyl ether. S2. Add the obtained quinoline triphenylamine iridium dichlorobridge [Ir(TPA)2Cl2]2 and 2-(2-pyridine)benzimidazole ligand to the reaction vessel, then add a solvent, preferably a mixture of dichloromethane and methanol (v:v=1:1). Under the condition of sufficient purging with inert gas N2, place the reaction vessel in the dark and reflux it. The reflux temperature is preferably 65-80℃, and the reaction time is preferably 6 h. After the reaction is completed 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 iridium metal coupling compound Ir-4. The preferred molar ratio of the quinoline triphenylamine iridium dichlorobridge [Ir(TPA)2Cl2]2 and the 2-(2-pyridine)benzimidazole ligand is 1:2; the preferred molar ratio of iridium metal coupling compound Ir-4 and potassium hexafluorophosphate is 1:5.

[0020] The synthetic route of this preparation method is shown below:

[0021]

[0022] The present invention also provides a nanoparticle prepared using the above-mentioned iridium metal coupling compound, wherein the particle size of the nanoparticle is preferably 100~200 nm.

[0023] The present invention also provides a method for producing nanoparticles, comprising: polyethylene glycol 2000MAL and arginine-glycine-aspartic peptide c (RGDfC) are dissolved in tetrahydrofuran and stirred for two hours. Then, a tetrahydrofuran solution of iridium metal conjugate Ir-4 is added to the mixture, followed by dropwise addition to ultrapure water. Stirring is preferably carried out at room temperature for 8 hours. Dialysis yields Ir-4@RNPs nanoparticles. The polyethylene glycol... 2000 The preferred mass ratio of MAL, c(RGDfC), and iridium coupling compound Ir-4 is 2:1:1.

[0024] 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.

[0025] The present invention also provides the application of the above-mentioned nanoparticles in the preparation of drugs for treating diseases by ultrasound thrombolysis.

[0026] 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.

[0027] Example 1 S1. IrCl3·3H2O (0.1 mmol, 0.0352 g) and quinoline triphenylamine ligand (0.3 mmol, 0.1116 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 N2 protection. 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 quinoline triphenylamine iridium dichlorobridge [Ir(TPA)2Cl2]2.

[0028] S2. Add the obtained quinoline triphenylamine iridium dichlorobridged [Ir(TPA)₂Cl₂]₂ (0.1 mmol, 0.1832 g) and 2-(2-pyridine)benzimidazole (0.2 mmol, 0.039 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 a full purging of inert gas N₂, the reaction is carried out in the dark under reflux at 80 °C for 6 h. After the reaction is complete and cooled to room temperature, add 200 mg of potassium hexafluorophosphate solid to the solution in the flask, and 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, Ir-4. The yield is 87%, and the molecular formula is C₂. 66H 47 IrN7 has a relative molecular mass of 1130.35 g / mol. Figure 1 The mass spectrometry data for the prepared Ir-4 shows a molecular weight of 1130.36 g / mol, which is the same as the fitted molecular weight, proving that the structure was successfully prepared.

[0029] 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 28 hours.

[0030] Example 3 The experimental conditions and steps were the same as in Example 1, except that in step S2, the reaction was stirred at 75°C for 6 hours.

[0031] The performance of the iridium metal coupling Ir-4 prepared in Example 1 was characterized in the following ways: 1. Preparation of nanoparticles 1 mg of polyethylene glycol 2000 -MAL and 0.5 mg c(RGDfC) were dissolved in tetrahydrofuran and stirred for two hours. Then, a tetrahydrofuran solution of 0.5 mg iridium metal conjugate Ir-4 was added to the mixture and added dropwise to 10 mL of ultrapure water. The mixture was stirred at room temperature for 8 hours and then dialyzed to obtain nanoparticles Ir-4@R NPs.

[0032] 2. Physical properties of Ir-4@R NPs Figure 1 The mass spectrometry data of Ir-4 is shown, which matches the theoretical values ​​to prove the structure. Figure 2 The image shows the particle size of Ir-4@R NPs (measured by a particle size potentiometer). The smaller image represents a transmission electron microscope (TEM) image of Ir-4@R NPs. As can be seen from the image, the particle size of Ir-4@R NPs in aqueous solution is 163.7 nm.

[0033] 3. Photophysical properties of Ir-4@R NPs The UV absorbance of Ir-4@R NPs in aqueous solution was measured using a UV-Vis spectrophotometer, and the results are as follows: Figure 3 As shown. Figure 3 This indicates that the maximum UV absorption of Ir-4@R NPs can reach around 550 nm.

[0034] Figure 4 The AIE performance test results of the Ir-4@R NPs prepared in this invention are shown in the figure, with tetrahydrofuran as a good solvent and water as a poor solvent. Figure 4 (a) shows the fluorescence emission patterns of Ir-4 at different water contents. Figure 4(b) shows the fluorescence emission curves of Ir-4 at different water contents. It can be seen that the Ir-4 prepared in this invention exhibits weak emission in pure tetrahydrofuran solution. The luminescence intensity increases with increasing water content, demonstrating its typical AIE characteristics. This will be beneficial for forming aggregated nanoparticles for subsequent diagnostic and therapeutic applications.

[0035] Figure 5 The Ir-4@R NPs prepared in this invention were subjected to in vitro singlet oxygen generation experiments under illumination and DPBF conditions using a UV spectrophotometer. The DPBF decrease curve at a specific nanometer and the degradation of DPBF under ultrasonic conditions demonstrate that Ir-4@RNPs have good singlet oxygen generation capabilities.

[0036] 4. Blood compatibility test of Ir-4@R NPs Figure 6 This figure shows the blood compatibility test data of the Ir-4@R NPs prepared in this invention. Different concentrations of Ir-4@RNPs were co-incubated with blood cells for 2 hours, and the supernatant was used for testing to calculate the hemolysis rate. As shown in the figure, the hemolysis rate of Ir-4@RNPs was less than 5%, indicating that Ir-4@R NPs have good blood compatibility and can be used for in vivo testing.

[0037] 5. In vitro thrombolysis experiment of Ir-4@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. Among them. 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-4@R NPs group reached 81.4% under ultrasound. The experimental results indicate that Ir-4@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).

[0038] 6. In vivo mouse experiments with Ir-4@R NPs Figure 8 In vivo chemiluminescence imaging of Ir-4@R NPs. Figure 8 (a) is a chemiluminescence signal image 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.

[0039] Figure 9 This is an in vivo thrombolysis assay of Ir-4@R NPs. 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. Ultrasound (1.0 W cm⁻¹) was used to visualize the thrombus. -2 Irradiating the thrombus site with 1MHz (1 MHz) has a good thrombolytic effect.

[0040] 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-4@R NPs. Figure 10 (b) The mouse organ sections were all normal, demonstrating that Ir-4@R NPs have good biocompatibility.

[0041] 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. An 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 iridium metal coupling according to claim 1, characterized in that, Includes the following steps: S1. Under nitrogen protection, IrCl3·3H2O and quinoline triphenylamine ligand were heated under reflux to obtain quinoline triphenylamine iridium dichlorobridge [Ir(TPA)2Cl2]2; S2. Under nitrogen protection, the quinoline triphenylamine iridium dichlorobridge and 2-(2-pyridine)benzimidazole ligand prepared in S1 were placed in the dark and refluxed in the presence of a solvent. After the reaction was completed, potassium hexafluorophosphate was added and the mixture was stirred to obtain the iridium metal conjugate Ir-4.

3. The method for preparing the above-mentioned iridium metal coupling according to claim 2, characterized in that, In step S2, the molar ratio of the quinoline triphenylamine iridium dichlorobridge and the 2-(2-pyridine)benzimidazole ligand is 1:

2.

4. The method for preparing the above-mentioned iridium metal coupling according to claim 2, characterized in that, In step S2, the reflux reaction temperature is 65~80℃, and the reflux reaction time is 6 h.

5. A nanoparticle, characterized in that, The nanoparticles were prepared using the iridium metal coupling compound as described in claim 1.

6. The method for preparing nanoparticles according to claim 5, characterized in that, include: polyethylene glycol 2000 -MAL and c(RGDfC) were dissolved in tetrahydrofuran and stirred. Then, a tetrahydrofuran solution of iridium metal coupling compound Ir-4 was added and mixed. The mixture was then added dropwise to ultrapure water and stirred. After dialysis, nanoparticles Ir-4@R NPs were obtained.

7. The method for preparing nanoparticles according to claim 6, characterized in that, The stirring temperature was room temperature, and the stirring time was 8 hours.

8. The method for preparing nanoparticles according to claim 6, characterized in that, The aforementioned polyethylene glycol 2000 The mass ratio of MAL, c(RGDfC) and iridium metal coupling is 2:1:

1.

9. The application of the nanoparticles as a sound therapy material according to claim 5.

10. The use of the nanoparticles according to claim 5 in the preparation of drugs for treating thrombotic diseases.

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