A bimetallic iridium complex conjugate, preparation method and application in thrombolysis

Abstract

The present invention belongs to the technical field of photodynamic therapy drugs, and specifically relates to a bimetallic iridium complex conjugate, a preparation method and its application in thrombolysis. The structural formula of the bimetallic iridium complex conjugate is shown below: By introducing Cu metal, the metal iridium complex has the property of chemiluminescence, so that it can simultaneously achieve the therapeutic effect of photothermal and photodynamic synergistic therapy. The bimetallic CuIr NPs provided by the present invention have good photodynamic and photothermal capabilities, which can overcome the problems of short excitation wavelength and limited light penetration depth required by the existing metal iridium complex itself. Experiments have shown that the material has a good therapeutic effect on thrombus, and it is promising to be developed as a phototherapy material and used as a thrombolytic photosensitizer.

Description

Technical Field

[0001] The present invention belongs to the technical field of photodynamic therapy drugs, and in particular relates to a bimetallic iridium complex conjugate, a preparation method and an application in thrombolysis. Technical Background

[0002] Phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), is an emerging, non-invasive, and real-time controllable light stimulation method with broad application prospects in disease treatment. Compared with traditional medications and surgery, phototherapy has inherent advantages such as non-invasiveness, low toxicity, simple operation, and rapid recovery, and has attracted much attention in the medical field.

[0003] Surgical removal of emboli or thrombolysis are the mainstays of clinical treatment for thrombotic diseases. However, current thrombolytic drugs often exhibit limited therapeutic efficacy due to their low selectivity, short half-life, and significant bleeding risk. In recent years, the non-invasive application of photosensitizers in the diagnosis and treatment of cardiovascular and cerebrovascular diseases has attracted widespread attention, particularly in the area of ​​thrombolysis, with the potential to become a next-generation therapeutic approach.

[0004] Although PTT has shown some potential in thrombolytic therapy, its nonspecific thermal damage to nearby tissues is a problem that limits its application. In addition, high cost, insufficient photothermal effect, low biodegradability and possible toxicity also limit the clinical transformation of PTT in thrombolytic therapy. PDT has been used in thrombolytic therapy research due to its advantages such as rapid onset, mild adverse reactions, and ability to be used in combination with other therapies. However, the clinical application of PDT is also limited by factors such as its dependence on oxygen and insufficient ROS generation efficiency of photosensitizers. Like PTT, it also has the problem of limited penetration depth due to insufficient light source wavelength.

[0005] As research deepens, the integration of PTT and PDT into a single system for synergistic treatment has shown promise as a highly effective and safe approach. PTT's oxygen-independence can offset PDT's oxygen-dependence, while the local thermal effect of PTT can accelerate local blood circulation and increase oxygen supply, promoting local PDT.

[0006] Transition metal materials are highly favored due to their rich excited state properties, such as high luminescence quantum efficiency, ultra-long luminescence lifetime, high luminescence stability, and multicolor luminescence. Compared with other transition metal complexes, cyclometallated iridium complexes have long triplet excitation lifetimes, large Stokes shifts, and outstanding photothermal generation capabilities, making them the most widely used class of transition metal complex materials. In 2020, Professor Gou Shaohua's team at Southeast University rationally designed a metal iridium complex with a donor-acceptor-donor (DAD) based on a simple skeleton structure for the first time, utilizing the synergistic effects of photodynamic and photothermal effects to achieve efficient anti-thrombotic therapeutic effects. However, the synthesis of this photosensitizer is relatively complex, which limits its widespread application. (AnIridium(III) Complex Bearing a Donor–Acceptor–Donor Type Ligand for NIR-Triggered Dual Phototherapy[J]. Advanced Functional Materials, 2020.). In 2021, the inventors rationally designed single-core and quadruple-core metal iridium complexes based on the skeleton structure of porphyrin and achieved excellent photothermal performance, but the fluorescent photosensitizer will affect the imaging effect due to its own background (Zhang Liping. Design, synthesis and phototherapy application research of deep red / near-infrared photosensitizers based on metal iridium complexes and organic small molecules [D]. Northeast Normal University, 2021.).

[0007] At present, iridium complexes suitable for integrated PTT and PDT therapy are still limited, and there are few reports on drug design targeting the thrombotic microenvironment. Providing more iridium complexes that can be used for integrated therapy is of great significance for the clinical selection of thrombolytic therapy. Summary of the Invention

[0008] In order to solve the above problems, one of the purposes of the present invention is to provide a bimetallic iridium complex conjugate, the structural formula of the bimetallic iridium complex conjugate is shown in formula (I):

[0009]

[0010] A second object of the present invention is to provide a method for preparing the bimetallic iridium complex conjugate as described above, comprising the following steps:

[0011] S1. Under nitrogen protection, IrCl3·3H2O and phenylpyridine ligand were heated under reflux to react to obtain phenylpyridine iridium dichloro bridge [Ir(ppy)2Cl2]2;

[0012] S2. Under nitrogen protection, the phenylpyridine iridium dichloride bridge and the porphyrin Schiff base ligand were placed in the dark under the action of a solvent and refluxed. After the reaction, potassium hexafluorophosphate was added at a molar ratio of 1:5 to the phenylpyridine iridium dichloride bridge and stirred continuously to obtain a binuclear metal iridium complex 2Ir;

[0013] S3. Under nitrogen protection, the binuclear iridium complex 2Ir was mixed with copper acetate, 15 mL 0.1 mmol DMF was added as a reaction solvent, and the mixture was stirred at 80° C. for 8 h to obtain a bimetallic iridium complex conjugate CuIr.

[0014] Preferably, in step S1, the molar ratio of IrCl3·3H2O to the phenylpyridine ligand is 1:(2.5-3).

[0015] Preferably, the solvent is 2-ethylene glycol ethyl ether.

[0016] Preferably, in step S1, the temperature of the reflux reaction is 120-130° C., and the time of the reflux reaction is 24-30 h.

[0017] Preferably, in step S2, the molar ratio of the phenylpyridine iridium dichloro bridge to the porphyrin Schiff base ligand is 1:1.

[0018] Preferably, in step S2, the temperature of the reflux reaction is 75-80° C., and the time of the reflux reaction is 8 hours.

[0019] Preferably, in step S2, the solvent is dichloromethane and methanol (v:v=1:1).

[0020] Preferably, in step S3, the molar ratio of 2Ir to copper acetate is 1:(3-5).

[0021] The synthetic route of the preparation method is shown as follows:

[0022]

[0023]

[0024] S1. To a reaction vessel containing a solvent and water, IrCl3·3H2O and a phenylpyridine ligand were added in a molar ratio of 1:(2.5-3), and the mixture was heated under reflux under nitrogen protection for 24-30 hours at 120-130°C. After the reaction was cooled to room temperature, a large amount of poor solvent water was added to precipitate the precipitate, which was filtered and the solvent was rinsed off with a large amount of water and ethanol. The resulting solid was dried to obtain phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2(L2);

[0025] The solvent in the reaction vessel is preferably 2-ethylene glycol ethyl ether.

[0026] S2. In a molar ratio of 1:1, the phenylpyridine iridium dichloro bridge [Ir(ppy)2Cl2]2 and porphyrin Schiff base ligand obtained above are added to the reaction vessel, and then a solvent is added, wherein the solvent is dichloromethane and methanol (v:v=1:1). When the inert gas N2 is fully filled, the reaction is placed in a dark place for reflux reaction. The reflux reaction temperature is 75-80°C and the reaction time is 8 hours. When the reaction is completed and cooled to room temperature, potassium hexafluorophosphate solid is added to the solution in the bottle, and stirring is continued at room temperature for 45-60 minutes. The solvent in the system is removed by a rotary evaporator, and then extracted with dichloromethane and water to remove excess potassium hexafluorophosphate solid. The obtained substance is washed with petroleum ether and dried, and purified by column chromatography to obtain a purple-red solid, namely 2Ir.

[0027] S3. Under nitrogen protection, 2Ir prepared in step S2 was mixed with copper acetate in a molar ratio of 1:(3-5), DMF was added as the reaction solvent, and the reaction was stirred at 80°C for 8 h. The obtained product was extracted with dichloromethane and water, and the solvent in the system was removed by rotary evaporation. The product was dried to obtain a red solid product, i.e., a bimetallic iridium complex conjugate CuIr.

[0028] The above preparation routes are mainly for explaining the present invention rather than limiting it.

[0029] A third object of the present invention is to provide a nanoparticle, which is prepared using the bimetallic iridium complex conjugate as described above, and has a particle size of 100 to 200 nm.

[0030] A fourth object of the present invention is to provide a method for preparing the nanoparticles as described above, comprising: dissolving CuIr and distearoylphosphatidylethanolamine-polyethylene glycol in tetrahydrofuran, adding the mixture dropwise to water, stirring and volatilizing the mixture at room temperature for 24 hours, and then dialyzing the mixture using a 2000 molecular weight dialysis membrane to obtain CuIr NPs with uniform particle size and stability.

[0031] A fifth object of the present invention is to provide the use of the bimetallic iridium complex conjugate or the nanoparticle as described above as a phototherapy material.

[0032] Preferably, the phototherapeutic material is a photoinitiator.

[0033] A sixth object of the present invention is to provide the use of the bimetallic iridium complex conjugate or the nanoparticles described above in the preparation of thrombolytic drugs.

[0034] Preferably, the thrombolytic drug is a drug for treating thrombosis.

[0035] The beneficial effects of the present invention are:

[0036] The present invention designs and synthesizes a bimetallic iridium complex conjugate CuIr in a simple and convenient way. By introducing Cu metal, the metal iridium complex has the property of chemiluminescence, enabling it to achieve the therapeutic effects of photothermal and photodynamic synergistic therapy at the same time.

[0037] The present invention also provides nanoparticles CuIr NPs formed by bio-encapsulating a bimetallic iridium complex conjugate CuIr into an amphiphilic polymer distearoylphosphatidylethanolamine-polyethylene glycol. The CuIr NPs have excellent bioluminescence properties and can specifically identify the location of the plug.

[0038] The CuIr NPs provided by the present invention can chemically react with excess ROS at the site of thrombus inflammation without the need for an external excitation light source, producing bioluminescence. Experiments have shown that they have a high penetration of 12 mm, overcoming the short excitation wavelength and limited penetration depth of existing metal iridium complexes. Test results show that CuIr NPs have excellent photodynamic and photothermal capabilities, indicating that this material has a strong therapeutic effect on thrombi and has the potential to be developed into a phototherapy material and used as a thrombolytic photosensitizer. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the mass spectrometry data of CuIr NPs prepared in Example 1 of the present invention.

[0040] Figure 2 This is the particle size potential diagram of CuIr NPs prepared in Example 1 of the present invention. In the figure, A, B, and C correspond to the particle sizes of TPP NPs, 2Ir NPs, and CuIr NPs, respectively; D is the potential of TPP NPs, 2Ir NPs, and CuIr NPs.

[0041] Figure 3 Ultraviolet absorption spectra (A in the figure) and fluorescence emission spectra (B in the figure) of CuIr NPs, 2Ir NPs, and TPP NPs prepared in Example 1 of the present invention in aqueous solution.

[0042] Figure 4 This is a diagram of ROS generation in aqueous solution of CuIr NPs (A in the figure), 2Ir NPs (B in the figure), and TPP NPs (C in the figure) prepared in Example 1 of the present invention with DPBF.

[0043] Figure 5The first-order kinetic curves of CuIr NPs, 2Ir NPs, and TPP NPs prepared in Example 1 of the present invention in aqueous solution (B in the figure is the logarithmic curve of A) show that a larger slope indicates more singlet oxygen is produced.

[0044] Figure 6 This is a photothermal data diagram of CuIr NPs, 2Ir NPs, and TPP NPs prepared in Example 1 of the present invention in aqueous solution, where A is the control group, B is the temperature change at different concentrations, C is the temperature change at different powers, and D is the photothermal cycle of the drug.

[0045] Figure 7 This is the thermal imaging data of CuIr NPs prepared in Example 1 of the present invention in aqueous solution.

[0046] Figure 8 This is a graph of the chemiluminescence properties of CuIr NPs prepared in Example 1 of the present invention. In the figure, A is the selectivity intensity diagram of different ROS, B is the chemiluminescence intensity at different times, C is the chemiluminescence intensity at different nm, D shows the concentration-dependent results, and E is the chemiluminescence penetration imaging under chicken breast coverage of different thicknesses.

[0047] Figure 9 This is an in vitro targeting test diagram of CuIr NPs prepared in Example 1 of the present invention.

[0048] Figure 10 This is a test chart of the antioxidant capacity of CuIr NPs prepared in Example 1 of the present invention, where A is the total antioxidant capacity test and B is the hydrogen peroxide consumption.

[0049] Figure 11 This is a hemolysis test diagram of CuIr NPs prepared in Example 1 of the present invention.

[0050] Figure 12 This is a graph showing the thrombolysis data of CuIr NPs prepared in Example 1 of the present invention under different conditions.

[0051] Figure 13 This is a photothermal imaging image of the thrombus location in mice induced by CuIr NPs prepared in Example 1 of the present invention.

[0052] Figure 14 This is a chemiluminescent imaging image of the thrombus location in mice using CuIr NPs prepared in Example 1 of the present invention.

[0053] Figure 15 This is a diagram of thrombolysis in mice by CuIr NPs prepared in Example 1 of the present invention.

[0054] Figure 16 This is the laser speckle pattern of CuIr NPs prepared in Example 1 of the present invention in mice. DETAILED DESCRIPTION

[0055] Unless otherwise specified, the terms used herein have the meanings commonly understood by those skilled in the art.

[0056] The technical solution of the present invention is described in more detail below with reference to the embodiments:

[0057] Example 1

[0058] Bimetallic iridium complex conjugate CuIr, the structural formula is shown in Formula I:

[0059]

[0060] The preparation method of the above-mentioned bimetallic iridium complex conjugate is:

[0061] S1. To a round-bottom flask containing 30 mL of 2-ethylene glycol ethyl ether and 10 mL of water, add IrCl3·3H2O (0.1 mmol, 0.0352 g) and phenylpyridine ligand (0.3 mmol, 0.0471 g). Under a nitrogen atmosphere, heat and reflux at 120°C for 24 h. After the reaction mixture cools to room temperature, add a large amount of water, a poor solvent, to precipitate the precipitate, which is filtered and washed multiple times with large amounts of water and ethanol. The resulting solid is dried to obtain phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2.

[0062] S2. To a 100 mL single-necked flask, add the phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2 (0.1 mmol, 0.1072 g) 1 and porphyrin Schiff base (0.1 mmol, 0.0822 g) obtained above, followed by 60 mL of dichloromethane and methanol (v:v = 1:1) as the solvent for the reaction system. Under full N2 inert gas, the reaction was placed in the dark and refluxed at 80°C for 8 h. After the reaction was completed and cooled to room temperature, 10 equivalents of potassium hexafluorophosphate solid was added to the solution in the flask. Stirring was continued at room temperature for 45 min. The solvent was removed from the system using a rotary evaporator, and the excess potassium hexafluorophosphate solid was removed by extraction with dichloromethane and water. The resulting material was washed with petroleum ether and dried. Purification by column chromatography yielded 2Ir as a purple-red solid.

[0063] S3. 2Ir (0.1 mmol, 0.1824 g) and copper acetate (0.5 mmol, 0.0998 g) were added to a 100 mL beaker, and DMF was used as the reaction solvent. The reaction was stirred at 80°C for 8 h. The product was extracted with dichloromethane and water. The solvent in the system was removed by rotary evaporation and dried to obtain a red solid product, namely, a bimetallic iridium complex conjugate CuIr, with a yield of 90%. The molecular formula is C 36H 22 IrN4Na2O4, relative molecular mass is 1885.42g / mol. Figure 1 The mass spectrometry data of the prepared CuIr showed a molecular weight of 942.43 g / mol, which was the same as the fitted molecular weight (CuIr has two charges and the molecular ion peak is 1 / 2 of the total mass), which can prove the structure.

[0064] Example 2

[0065] S1. To a round-bottom flask containing 30 mL of 2-ethylene glycol ethyl ether and 10 mL of water, add IrCl3·3H2O (0.1 mmol, 0.0352 g) and phenylpyridine ligand (0.25 mmol, 0.0392 g). Under a nitrogen atmosphere, heat and reflux at 120°C for 24 h. After the reaction mixture cools to room temperature, add a large amount of water, a poor solvent, to precipitate the precipitate, which is filtered and washed several times with large amounts of water and ethanol. The resulting solid is dried to obtain phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2.

[0066] S2. To a 100 mL single-necked flask, add the phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2 (0.1 mmol, 0.1072 g) 1 and porphyrin Schiff base (0.1 mmol, 0.0822 g) obtained above, followed by 60 mL of dichloromethane and methanol (v:v = 1:1) as the solvent for the reaction system. Under full N2 inert gas, the reaction was placed in the dark and refluxed at 80°C for 8 h. After the reaction was completed and cooled to room temperature, 10 equivalents of potassium hexafluorophosphate solid was added to the solution in the flask. Stirring was continued at room temperature for 45 min. The solvent was removed from the system using a rotary evaporator, and the excess potassium hexafluorophosphate solid was removed by extraction with dichloromethane and water. The resulting material was washed with petroleum ether and dried. Purification by column chromatography yielded 2Ir as a purple-red solid.

[0067] S3. 2Ir (0.1 mmol, 0.1824 g) and copper acetate (0.5 mmol, 0.0998 g) were added to a 100 mL beaker, and DMF was used as the reaction solvent. The reaction was stirred at 80°C for 8 h. The product was extracted with dichloromethane and water. The solvent in the system was removed by rotary evaporation and dried to obtain a red solid product, namely, a bimetallic iridium complex conjugate CuIr, with a yield of 85%. The molecular formula is C 36 H 22 IrN4Na2O4, relative molecular mass is 1885.42g / mol.

[0068] Example 3

[0069] S1. To a round-bottom flask containing 30 mL of 2-ethylene glycol ethyl ether and 10 mL of water, add IrCl3·3H2O (0.1 mmol, 0.0352 g) and phenylpyridine ligand (0.3 mmol, 0.0471 g). Under a nitrogen atmosphere, heat and reflux at 120°C for 30 h. After the reaction mixture cools to room temperature, a large amount of water, a poor solvent, is added to precipitate the precipitate, which is filtered and washed multiple times with large amounts of water and ethanol. The resulting solid is dried to obtain phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2.

[0070] S2. To a 100 mL single-necked flask, add the phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2 (0.1 mmol, 0.1072 g) 1 and porphyrin Schiff base (0.1 mmol, 0.0822 g) obtained above, followed by 60 mL of dichloromethane and methanol (v:v = 1:1) as the solvent for the reaction system. Under full N2 inert gas, the reaction was placed in the dark and refluxed at 75°C for 8 h. After the reaction was completed and cooled to room temperature, 10 equivalents of potassium hexafluorophosphate solid was added to the solution in the flask. Stirring was continued at room temperature for 45 min. The solvent was removed from the system using a rotary evaporator, and the excess potassium hexafluorophosphate solid was removed by extraction with dichloromethane and water. The resulting material was washed with petroleum ether and dried. Purification by column chromatography yielded 2Ir as a purple-red solid.

[0071] S3. 2Ir (0.1 mmol, 0.1824 g) and copper acetate (0.5 mmol, 0.0998 g) were added to a 100 mL beaker, and DMF was used as the reaction solvent. The reaction was stirred at 80°C for 8 h. The product was extracted with dichloromethane and water. The solvent in the system was removed by rotary evaporation and dried to obtain a red solid product, namely, the bimetallic iridium complex conjugate CuIr, with a yield of 93%. The molecular formula is C 36 H 22 IrN4Na2O4, relative molecular mass is 1885.42g / mol.

[0072] Example 4

[0073] S1. To a round-bottom flask containing 30 mL of 2-ethylene glycol ethyl ether and 10 mL of water, add IrCl3·3H2O (0.1 mmol, 0.0352 g) and phenylpyridine ligand (0.3 mmol, 0.0471 g). Under a nitrogen atmosphere, heat and reflux at 130°C for 24 h. After the reaction mixture cools to room temperature, add a large amount of water, a poor solvent, to precipitate the precipitate, which is filtered and washed multiple times with large amounts of water and ethanol. The resulting solid is dried to obtain phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2.

[0074] S2. To a 100 mL single-necked flask, add the phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2 (0.1 mmol, 0.1072 g) 1 and porphyrin Schiff base (0.1 mmol, 0.0822 g) obtained above, followed by 60 mL of dichloromethane and methanol (v:v = 1:1) as the solvent for the reaction system. Under full N2 inert gas, the reaction was placed in the dark and refluxed at 75°C for 8 h. After the reaction was completed and cooled to room temperature, 10 equivalents of potassium hexafluorophosphate solid was added to the solution in the flask. Stirring was continued at room temperature for 45 min. The solvent was removed from the system using a rotary evaporator, and the excess potassium hexafluorophosphate solid was removed by extraction with dichloromethane and water. The resulting material was washed with petroleum ether and dried. Purification by column chromatography yielded 2Ir as a purple-red solid.

[0075] S3. 2Ir (0.1 mmol, 0.1824 g) and copper acetate (0.5 mmol, 0.0998 g) were added to a 100 mL beaker, and DMF was used as the reaction solvent. The reaction was stirred at 80°C for 8 h. The product was extracted with dichloromethane and water. The solvent in the system was removed by rotary evaporation and dried to obtain a red solid product, namely, the bimetallic iridium complex conjugate CuIr, with a yield of 91%. The molecular formula is C 36 H 22 IrN4Na2O4, relative molecular mass is 1885.42g / mol.

[0076] Example 5

[0077] S1. To a round-bottom flask containing 30 mL of 2-ethylene glycol ethyl ether and 10 mL of water, add IrCl3·3H2O (0.1 mmol, 0.0352 g) and phenylpyridine ligand (0.3 mmol, 0.0471 g). Under a nitrogen atmosphere, heat and reflux at 120°C for 24 h. After the reaction mixture cools to room temperature, add a large amount of water, a poor solvent, to precipitate the precipitate, which is filtered and washed multiple times with large amounts of water and ethanol. The resulting solid is dried to obtain phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2.

[0078] S2. To a 100 mL single-necked flask, add the phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2 (0.1 mmol, 0.1072 g) 1 and porphyrin Schiff base (0.1 mmol, 0.0822 g) obtained above, followed by 60 mL of dichloromethane and methanol (v:v = 1:1) as the solvent for the reaction system. Under full N2 inert gas, the reaction was placed in the dark and refluxed at 80°C for 8 h. After the reaction was completed and cooled to room temperature, 10 equivalents of potassium hexafluorophosphate solid was added to the solution in the flask. Stirring was continued at room temperature for 45 min. The solvent was removed from the system using a rotary evaporator, and the excess potassium hexafluorophosphate solid was removed by extraction with dichloromethane and water. The resulting material was washed with petroleum ether and dried. Purification by column chromatography yielded 2Ir as a purple-red solid.

[0079] S3. 2Ir (0.1 mmol, 0.1824 g) and copper acetate (0.3 mmol, 0.0599 g) were added to a 100 mL beaker, and DMF was used as the reaction solvent. The reaction was stirred at 80°C for 8 h. The product was extracted with dichloromethane and water. The solvent in the system was removed by rotary evaporation and dried to obtain a red solid product, namely, the bimetallic iridium complex conjugate CuIr, with a yield of 88%. The molecular formula is C 36 H 22 IrN4Na2O4, relative molecular mass is 1885.42g / mol.

[0080] Example 6

[0081] S1. To a round-bottom flask containing 30 mL of 2-ethylene glycol ethyl ether and 10 mL of water, add IrCl3·3H2O (0.1 mmol, 0.0352 g) and phenylpyridine ligand (0.3 mmol, 0.0471 g). Under a nitrogen atmosphere, heat and reflux at 120°C for 24 h. After the reaction mixture cools to room temperature, add a large amount of water, a poor solvent, to precipitate the precipitate, which is filtered and washed multiple times with large amounts of water and ethanol. The resulting solid is dried to obtain phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2.

[0082] S2. To a 100 mL single-necked flask, add the phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2 (0.1 mmol, 0.1072 g) 1 and porphyrin Schiff base (0.1 mmol, 0.0822 g) obtained above, followed by 60 mL of dichloromethane and methanol (v:v = 1:1) as the solvent for the reaction system. Under full N2 inert gas, the reaction was placed in the dark and refluxed at 80°C for 8 h. After the reaction was completed and cooled to room temperature, 10 equivalents of potassium hexafluorophosphate solid was added to the solution in the flask. Stirring was continued at room temperature for 45 min. The solvent was removed from the system using a rotary evaporator, and the excess potassium hexafluorophosphate solid was removed by extraction with dichloromethane and water. The resulting material was washed with petroleum ether and dried. Purification by column chromatography yielded 2Ir as a purple-red solid.

[0083] S3. 2Ir (0.1 mmol, 0.1824 g) and copper acetate (0.4 mmol, 0.080 g) were added to a 100 mL beaker, and DMF was used as the reaction solvent. The reaction was stirred at 77°C for 8 h. The product was extracted with dichloromethane and water. The solvent in the system was removed by rotary evaporation and dried to obtain a red solid product, namely, the bimetallic iridium complex conjugate CuIr, with a yield of 86%. The molecular formula is C 36 H 22 IrN4Na2O4, relative molecular mass is 1885.42g / mol.

[0084] experiment

[0085] The performance of the bimetallic iridium complex conjugate CuIr prepared in Example 1 was characterized as follows:

[0086] 1. Preparation of Nanoparticles

[0087] CuIr (0.0010 g, prepared in Example 1) and distearoylphosphatidylethanolamine-polyethylene glycol (0.0020 g) were dissolved in 1 ml of tetrahydrofuran, and then added dropwise to 10 ml of water. The mixture was stirred and evaporated overnight at room temperature. After evaporation, the mixture was dialyzed using a 2000 molecular weight dialysis membrane to obtain CuIr NPs with uniform and stable particle size.

[0088] 2Ir (0.0010 g) and distearoylphosphatidylethanolamine-polyethylene glycol (0.0020 g) were dissolved in 1 ml of tetrahydrofuran, respectively, and then added dropwise to the prepared 10 mL of water. The mixture was stirred and evaporated overnight at room temperature. After evaporation, it was dialyzed using a 2000 molecular weight dialysis membrane to obtain 2IrNPs with uniform particle size and stable dispersion in the aqueous solution.

[0089] TPP (0.0010 g) and distearoylphosphatidylethanolamine-polyethylene glycol (0.0020 g) were dissolved in 1 ml of tetrahydrofuran respectively, and then added dropwise to the prepared 10 mL of water. The mixture was stirred and evaporated overnight at room temperature. After evaporation, the mixture was dialyzed using a 1000 molecular weight dialysis membrane to obtain TPPNPs with uniform particle size and stable dispersion in the aqueous solution.

[0090] 2. Physical properties of CuIr NPs

[0091] Figure 2 The particle size and potential diagram of TPP NPs, 2Ir NPs, and CuIr NPs (measured by particle size potential analyzer). It can be seen from the figure that the particle sizes of TPP NPs, 2Ir NPs, and CuIr NPs in aqueous solution are 75.91nm, 92.66nm, and 129nm, respectively. At the same time, the potential of CuIr NPs was measured to be -25mV.

[0092] The uniform particle size of <200 nm indicates that CuIr has good biocompatibility and a negative potential, indicating that it can have a good circulation effect in the body.

[0093] 3. Photophysical properties of CuIr NPs

[0094] The UV absorption and fluorescence emission of CuIr NPs in aqueous solution were measured by UV-visible spectrophotometer and fluorescence spectrometer. Figure 3 As shown in the figure, the absorption in the ultraviolet absorption disappears because the Cu metal enters the porphyrin core, and the two original Q bands of the porphyrin disappear. The emission intensity becomes weaker due to the introduction of the Ir core, and the fluorescence is quenched after the introduction of Cu.

[0095] Figure 4 、 Figure 5 For the CuIr NPs prepared in the present invention, an in vitro singlet oxygen generation experiment and related kinetic curves under the conditions of light and DPBF were measured by ultraviolet spectrophotometer. The degradation of DPBF under light proved that CuIr NPs had good singlet oxygen generation ability.

[0096] 4. Photothermal properties of CuIr NPs

[0097] Thermocouple photothermal data, Figure 6 Middle A shows that compared with the control group H2O, the drug groups (TPP NPs, 2Ir NPs, CuIr NPs) have better photothermal generation capabilities; Figure 6 Middle B shows that the higher the concentration of CuIr NPs, the higher the temperature under the same illumination time, indicating that it is concentration-dependent; Figure 6Middle C shows that CuIr NPs generate heat at different powers. The stronger the power, the higher the temperature rise, indicating that it is power-dependent. Figure 6 D in the middle shows the temperature change of CuIr NPs after five heating and cooling cycles, which shows that the drug has good photothermal cycling ability.

[0098] Figure 7 This is the thermal imaging image of CuIr NPs corresponding to the photothermal data. Under illumination conditions, the solution temperature increases with the extension of illumination time.

[0099] 5. Chemiluminescence experiment of CuIr NPs

[0100] Figure 8 The relevant data table of CuIr NPs chemiluminescence was used to test the relevant chemiluminescence using IVIS mouse in vivo imaging system. Figure 8 In middle A, different types of RONS were co-incubated with CuIrNPs in a 96-well plate in response to different ROS, ONOO - The luminescence intensity was the strongest, and ONOO - As a marker substance, it can react with CuIr NPs to produce chemiluminescence, illuminating the inflammatory site. Figure 8 In Figure B, at different illumination times, the test results show that the drug luminescence intensity is highest when the illumination is 90s, and this time is selected as the illumination time for in vivo drug imaging. Figure 8 In middle C, the dotted line graph of chemiluminescence intensity at different nanometers shows that the drug has the highest intensity of bioluminescence emission at 800 nm. Figure 8 Middle D, different proportions of ONOO - The results of coexistence measurements with the drug showed a linear correlation. Figure 8 In Figure E, chicken breasts of varying thicknesses are coated on the drug. The chemiluminescence can penetrate up to 12 mm, a depth that facilitates imaging of drugs deep within inflammatory sites. This offers significant advantages over photosensitizers that rely on short-wavelength excitation light.

[0101] Figure 9 In vitro targeting testing of the CuIr NPs of the present invention was performed. Fresh mouse blood, without anticoagulant, was added to a 5 mL test tube and refrigerated at 4°C for 24 hours to produce thrombi. The drug was then incubated with the thrombi, and the chemiluminescence intensity was measured at 2, 4, and 6 hours. The results showed that longer incubation time with the drug increased the chemiluminescence intensity, demonstrating the drug's excellent targeting ability for thrombi.

[0102] 6. Antioxidant properties of CuIr NPs

[0103] Figure 10The test results of the antioxidant capacity of CuIr NPs are shown in Figure 2. Figure 10 Figure A, using the kit for detection, shows that when the concentration of CuIr NPs reaches 300μg / mL, the consumption rate of DPPH can reach 70%. Figure 10 In panel B, co-incubation with hydrogen peroxide detected a consumption rate of nearly 60%.

[0104] At the thrombus site, the hydrogen peroxide content is far higher than that of normal tissue. The consumption of hydrogen peroxide will effectively improve the microenvironment at the thrombus site. At the same time, it proves that CuIr NPs have good antioxidant capacity, showing that it has a good therapeutic effect on inflammation in the body.

[0105] 7. Hemolysis experiment of CuIr NPs

[0106] Figure 11 CuIr NPs hemolysis test. Different concentrations of CuIr NPs were incubated with red blood cells in hot water at 37°C for 2 hours. The PBS group served as the negative control, and the water group served as the positive control. The hemolysis rate of the drug at 200 μg / mL remained below 5%, indicating good biosafety.

[0107] 8. In vitro thrombolysis experiments of CuIr NPs

[0108] 5 mL of NPs from different groups and thrombus were placed in 10 mL vials for in vitro thrombolysis test. The results were as follows: Figure 12 As shown in the figure, compared with the control group, the CuIr NPs group was able to simultaneously activate PDT / PTT dual-mode thrombolysis under laser irradiation, achieving a thrombolysis rate of 68.7%. The study also demonstrated that both PDT and PTT during thrombolysis were combined, with results showing that only PTT achieved a thrombolysis rate of 34.7%, exceeding the efficiency of PDT using low-temperature thrombolysis. These experimental results demonstrate that CuIr NPs possess excellent in vitro thrombolytic activity.

[0109] 9. In vivo experiments on mice using CuIr NPs

[0110] Figure 13 In vivo photothermal imaging of CuIr NPs was performed. Mice were anesthetized, the thoracotomy was performed, and the right carotid artery was located. The artery was then incubated with a 10% FeCl₃ solution for approximately 10 minutes, causing the vessel to darken and thrombus formation to be complete. Following tail vein administration, the drug circulated to the thrombus site, creating a thrombus. An infrared thermal imager was used to record the temperature of the thrombus site as the drug accumulated within the thrombus over time. The results showed that the temperature at the thrombus site gradually increased with increasing illumination time.

[0111] Figure 14In vivo chemiluminescence imaging of CuIr NPs shows that after tail vein administration, the drug circulates to the thrombus over time, with pronounced chemiluminescence under illumination. Maximum aggregation is achieved at 60 minutes, signaling the start of thrombolytic therapy.

[0112] Figure 15 Figure 1 shows an in vivo thrombolytic test of CuIr NPs. Mice were anesthetized, the thoracotomy was performed, and the right carotid artery was located. The artery was then incubated with 10% FeCl₃ solution for approximately 10 minutes, causing the vessel to turn black. After thrombus formation was complete, the drug was circulated to the thrombus site. Laser (635nm 0.8w / cm 2 ) irradiated the thrombus location, and the thrombolysis rate in the body reached 72%, which had a good thrombolytic effect.

[0113] Figure 16 This image shows blood flow recorded during laser speckle pattern monitoring of thrombolysis using CuIr NPs. After a thrombus formed in vivo, the thrombolysis rate in the CuIr NPs group increased with prolonged illumination, with the thrombus gradually dissolving and blood flow gradually resuming. Unlike the common thrombolytic drug UKase, which can cause secondary embolism after thrombus clearance, leading to further reductions in blood flow, the CuIr NPs-treated group experienced no re-embolism during thrombolysis. This demonstrates the excellent PDT / PTT capabilities of CuIr NPs, which can effectively prevent re-embolism and perform well in in vivo treatment.

[0114] 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 above 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 in the scope of protection of the present invention.

Claims

1. A bimetallic iridium complex conjugate, characterized in that: The structural formula is shown in formula (I):

2. A method for preparing a bimetallic iridium complex conjugate according to claim 1, characterized in that: The following steps are involved: S1. Under nitrogen protection, IrCl3·3H2O and phenylpyridine ligand were heated under reflux to react to obtain phenylpyridine iridium dichloro bridge; S2. Under nitrogen protection, the phenylpyridine iridium dichloride bridge and the porphyrin Schiff base ligand were placed in the dark under the action of a solvent and refluxed. After the reaction, potassium hexafluorophosphate was added at a molar ratio of 1:5 to the phenylpyridine iridium dichloride bridge and stirred continuously to obtain a binuclear metal iridium complex 2Ir; S3. Under nitrogen protection, the binuclear metal iridium complex 2Ir was mixed with copper acetate, DMF was added as a reaction solvent, and the mixture was stirred at 80° C. for 8 h to react to obtain a bimetallic iridium complex conjugate.

3. The method for preparing a bimetallic iridium complex conjugate according to claim 2, wherein: In step S1, the molar ratio of IrCl3·3H2O to phenylpyridine ligand is 1:(2.5-3).

4. The method for preparing a bimetallic iridium complex conjugate according to claim 2, wherein: In step S1, the temperature of the reflux reaction is 120-130° C., and the time of the reflux reaction is 24-30 h.

5. The method for preparing a bimetallic iridium complex conjugate according to claim 2, wherein: In step S2, the molar ratio of the phenylpyridine iridium dichloro bridge and the porphyrin Schiff base ligand is 1:1, the solvent is a mixture of dichloromethane and methanol in a volume ratio of 1:1, the reflux reaction temperature is 75-80° C., and the reflux reaction time is 8 hours.

6. The method for preparing a bimetallic iridium complex conjugate according to claim 2, wherein: In step S3, the molar ratio of 2Ir to copper acetate is 1:(3-5).

7. A nanoparticle, characterized in that: The nanoparticles are prepared using the bimetallic iridium complex conjugate according to claim 1, and the particle size of the nanoparticles is 100 to 200 nm.

8. A method for preparing the nanoparticles according to claim 7, characterized in that: The method is as follows: 1 mg of bimetallic iridium complex conjugate and 2 mg of distearoylphosphatidylethanolamine-polyethylene glycol were dissolved in 1 mL of tetrahydrofuran respectively, added dropwise to 10 mL of water, stirred and evaporated at room temperature for 24 hours, and then dialyzed using a 2000 molecular weight dialysis membrane to obtain uniform and stable nanoparticles CuIr NPs.

9. Use of the bimetallic iridium complex conjugate according to claim 1 or the nanoparticles according to claim 7 in the preparation of phototherapy materials.

10. The use according to claim 9, wherein the phototherapy material is a photosensitive initiator.

11. Use of the bimetallic iridium complex conjugate according to claim 1 or the nanoparticles according to claim 7 in the preparation of thrombolytic drugs.

12. The use according to claim 11, wherein the thrombolytic drug is a drug for treating thrombosis.

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