A bimetallic iridium complex conjugate, nanoparticles, preparation method and application in ischemic stroke

Abstract

The present invention provides a bimetallic iridium complex conjugate, nanoparticles, a preparation method and an application in ischemic stroke, belonging to the technical field of photodynamic therapy drugs. The structural formula of the bimetallic iridium complex conjugate is shown in formula (I). The present invention also provides a method for preparing the above-mentioned bimetallic iridium complex conjugate. The present invention also provides a nanoparticle. The bimetallic iridium complex prepared by the present invention has the property of chemiluminescence, so that it can simultaneously achieve the synergistic thrombolytic therapeutic effect of photothermal and photodynamic therapy, and its antioxidant property has a significant effect on inhibiting nerve cell death and saving the infarcted brain damage site. The conjugate provided by the present invention has good photodynamic and photothermal capabilities, which can overcome the problems of the 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 ischemic stroke.

Description

Technical Field

[0001] The present invention belongs to the technical field of photodynamic therapy drugs, and specifically relates to a bimetallic iridium complex conjugate, nanoparticles, a preparation method and application in ischemic stroke. Background Art

[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 drug thrombolysis for ischemic stroke 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] Ischemic stroke (IS) is caused by insufficient blood and oxygen supply to the brain and is the second leading cause of death worldwide and a major cause of disability. In addition, thrombotic ischemic stroke accounts for more than 80% of all stroke events. Therapies such as thrombolysis and thrombectomy are crucial for effectively restoring blood supply to the penumbra, and timely reperfusion can effectively rescue damaged neurons by restoring coronary blood flow to ischemic tissue. It is worth noting that high-dose thrombolytic drugs are often used to effectively restore blood flow. However, the use of these drugs inevitably damages the integrity of the blood-brain barrier (BBB), thereby increasing the risk of intracranial hemorrhage. Reperfusion also leads to a significant increase in blood oxygen levels in the infarcted area, thereby releasing excessive cytotoxic reactive oxygen and nitrogen species (RONS) (such as peroxynitrite (ONOO - )) reacts with proteins or nucleotides, destroys DNA structure, and induces nerve cell death. Therefore, it is possible to use specific recognition of ONOO - Near-infrared probes to accurately monitor ONOO - levels, thus facilitating early diagnosis of ischemic stroke. This approach is highly beneficial in mitigating the adverse effects of neuronal death on brain function, such as motor impairment and impaired consciousness. Therefore, a comprehensive treatment approach is urgently needed for ischemic stroke that can safely achieve thrombolysis, monitor and eliminate RONS, and salvage the ischemic penumbra near the infarct core, thereby minimizing the final infarct size and improving neurological deficits.

[0005] 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.

[0006] 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.

[0007] 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.).

[0008] At present, iridium complexes suitable for integrated therapy of ischemic stroke 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 stroke treatment. Summary of the Invention

[0009] The purpose of the present invention is to provide a bimetallic iridium complex conjugate, nanoparticles, a preparation method and application in ischemic stroke. The metal iridium complex of the present invention has chemiluminescent properties, which can realize visual monitoring in the early development stage of the disease. At the same time, its excellent antioxidant properties have excellent therapeutic effects on saving the ischemic penumbra and reducing the area of ​​cerebral infarction.

[0010] The present invention first provides a bimetallic iridium complex conjugate, the structural formula of the bimetallic iridium complex conjugate is shown in formula (I):

[0011]

[0012] The present invention also provides a method for preparing the above-mentioned bimetallic iridium complex conjugate, comprising the following steps:

[0013] 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;

[0014] S2. Under nitrogen protection, the phenylpyridine iridium dichloro bridge and porphyrin Schiff base ligand prepared in S1 were placed in the dark under the action of a solvent and refluxed. After the reaction, potassium hexafluorophosphate was added and stirred to obtain a tetranuclear iridium complex 4Ir.

[0015] S3. Under nitrogen protection, the tetranuclear metal iridium complex 4Ir prepared in S2 is mixed with manganese chloride and reacted to obtain a bimetallic iridium complex conjugate 4IrMn.

[0016] Preferably, in step S2, the molar ratio of the phenylpyridine iridium dichloro bridge to the porphyrin Schiff base ligand is 1:1.

[0017] Preferably, in step S2, the temperature of the reflux reaction is 75-80° C., and the reflux reaction time is 8 h.

[0018] Preferably, in step S3, the molar ratio of the tetranuclear metal iridium complex 4Ir to manganese chloride is 1:(5-7).

[0019] Preferably, in step S3, the reaction temperature is 75° C.-80° C., and the reaction time is 9-12 h.

[0020] The present invention also provides a nanoparticle, which is prepared by adopting the above-mentioned bimetallic iridium complex conjugate, and the particle size of the nanoparticle is 100-200 nm.

[0021] The present invention also provides a method for producing nanoparticles, comprising:

[0022] The bimetallic iridium complex conjugate 4IrMn and distearoylphosphatidylethanolamine-polyethylene glycol were dissolved in solvents respectively, and the dissolved solutions were added dropwise into water. The mixture was stirred and evaporated at room temperature, and then dialyzed to obtain nanoparticles 4IrMnNPs.

[0023] The present invention also provides application of the nanoparticles as phototherapy materials.

[0024] The present invention also provides use of the nanoparticles in preparing a drug for treating ischemic stroke.

[0025] Beneficial effects of the present invention

[0026] The present invention provides a bimetallic iridium complex conjugate, nanoparticles, a preparation method and an application in ischemic stroke. The present invention designs and synthesizes a bimetallic iridium complex conjugate 4IrMn in a simple and convenient manner. The synthesized metal iridium complex has chemiluminescence properties, which enables visual monitoring in the early stages of disease development. At the same time, its excellent antioxidant properties have excellent therapeutic effects on saving the ischemic penumbra and reducing the area of ​​cerebral infarction.

[0027] The present invention also provides nanoparticles 4IrMn NPs formed by bio-encapsulating a bimetallic iridium complex conjugate 4IrMn into an amphiphilic polymer distearoylphosphatidylethanolamine-polyethylene glycol. The 4IrMn NPs have excellent bioluminescent properties and can specifically identify the location of reperfusion.

[0028] The 4IrMn NPs provided by this invention react chemically with excess ROS at the site of inflammation, producing bioluminescence without the need for an external excitation light source. Experiments have shown that they have a high penetration of 10 mm, overcoming the short excitation wavelength and limited penetration depth of existing iridium complexes. Test results also show that CuIr NPs possess excellent antioxidant capacity, suggesting that this material has a promising therapeutic effect against stroke and holds promise for development as a phototherapy material and as a stroke treatment drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Mass spectrometry data of 4IrMnNPs prepared in Example 1 of the present invention.

[0030] Figure 2 This is a particle size potential diagram of 4IrMnNPs prepared in Example 1 of the present invention, where the particle sizes and potentials of TPP NPs, IrNPs, IrMn NPs, 4IrNPs, and 4IrMn NPs are respectively corresponding.

[0031] Figure 3These are the ultraviolet absorption spectra and fluorescence emission spectra of TPP NPs, IrNPs, IrMn NPs, 4IrNPs and 4IrMn NPs prepared in Example 1 of the present invention in aqueous solution.

[0032] Figure 4 The first-order kinetic curves of TPP NPs, IrNPs, IrMn NPs, 4IrNPs and 4IrMn NPs in aqueous solution prepared in Example 1 of the present invention (B in the figure is the logarithmic curve of A) show that a larger slope indicates more singlet oxygen is produced.

[0033] Figure 5 This is a photothermal data diagram of TPP NPs, IrNPs, IrMn NPs, 4IrNPs, and 4IrMn NPs prepared in Example 1 of the present invention in aqueous solution, including the control group, temperature changes at different concentrations, temperature changes at different powers, and the photothermal cycle of the drug.

[0034] Figure 6 This is the thermal imaging data of 4IrMnNPs prepared in Example 1 of the present invention in aqueous solution.

[0035] Figure 7 This is a graph of the chemiluminescent properties of 4IrMn NPs prepared in Example 1 of the present invention, including the selectivity intensity graph of different RONS, chemiluminescence intensity at different times, and chemiluminescence penetration imaging of chemiluminescence intensity at different nm under chicken breast coverage of different thicknesses.

[0036] Figure 8 This is an in vitro targeting test diagram of 4IrMnNPs prepared in Example 1 of the present invention.

[0037] Figure 9 This is a graph showing the antioxidant capacity test of 4IrMn NPs prepared in Example 1 of the present invention, including the total antioxidant capacity test and hydrogen peroxide consumption.

[0038] Figure 10 This is a graph showing the thrombolysis data of 4IrMnNPs prepared in Example 1 of the present invention under different conditions.

[0039] Figure 11 This is a test chart of the intracellular antioxidant capacity of 4IrMnNPs prepared in Example 1 of the present invention.

[0040] Figure 12 This is a graph showing the chemiluminescence monitoring test of thrombosis in mice using 4IrMnNPs prepared in Example 1 of the present invention.

[0041] Figure 13 Figure 1 shows the thrombolysis data of mice under different conditions using 4IrMnNPs prepared in Example 1 of the present invention.

[0042] Figure 14 This is a chemiluminescent imaging image of the stroke location in the mouse brain of 4IrMn NPs prepared in Example 1 of the present invention.

[0043] Figure 15 This is a TTC staining image of brain slices of rats treated with 4IrMn NPs in Example 1 of the present invention.

[0044] Figure 16 This is a behavioral monitoring chart of 4IrMnNPs prepared in Example 1 of the present invention for the treatment of stroke in mice. DETAILED DESCRIPTION

[0045] The present invention first provides a bimetallic iridium complex conjugate, the structural formula of the bimetallic iridium complex conjugate is shown in formula (I):

[0046]

[0047] The present invention also provides a method for preparing the above-mentioned bimetallic iridium complex conjugate, comprising the following steps:

[0048] S1. To a reaction vessel containing a solvent and water, IrCl3·3H2O and a phenylpyridine ligand are added, and the reaction is heated under reflux under nitrogen protection, preferably at 120-130°C for 24-30h, and the reaction is cooled to room temperature, and then a large amount of poor solvent water is added thereto to precipitate the precipitate and filter it, and the solvent is rinsed with a large amount of water and ethanol. The resulting solid is dried to obtain phenylpyridine iridium dichloro bridge [Ir(ppy)2Cl2]2 (L2); the molar ratio of IrCl3·3H2O to phenylpyridine is preferably 1:(2.5-3), and the solvent is preferably 2-ethylene glycol ethyl ether;

[0049] S2. Add the above-obtained phenylpyridine iridium dichloro bridge [Ir(ppy)2Cl2]2 and porphyrin Schiff base ligand to the reaction vessel, and then add a solvent, wherein the solvent is preferably a mixture of 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 temperature of the reflux reaction is preferably 75-80°C, and the reaction time is preferably 8h. When the reaction is completed and cooled to room temperature, potassium hexafluorophosphate solid is added to the solution in the bottle, and the mixture is continued at room temperature. Stirring, the stirring time is preferably 45 to 60 minutes, using a rotary evaporator to remove the solvent in the system, then extracting with dichloromethane and water to remove excess potassium hexafluorophosphate solid, and washing the obtained substance with petroleum ether and drying, and purifying by column chromatography to obtain a purple-red solid, namely the tetranuclear metal iridium complex 4Ir; the molar ratio of the phenylpyridine iridium dichloro bridge [Ir(ppy)2Cl2]2 and the porphyrin Schiff base ligand is 1:1; the molar ratio of the phenylpyridine iridium dichloro bridge and potassium hexafluorophosphate is preferably 1:5.

[0050] S3. Under nitrogen protection, the tetranuclear metal iridium complex 4Ir prepared in step S2 is mixed with manganese chloride in a molar ratio of 1:(5-7), and DMF is added as a reaction solvent. The reaction is preferably carried out at 75°C-80°C for 9-12 hours, more preferably at 80°C for 9 hours. The obtained product is extracted with dichloromethane and water, and the solvent in the system is removed by a rotary evaporator. The product is dried to obtain a purple-black solid product, namely, the bimetallic iridium complex conjugate 4IrMn.

[0051] The synthetic route of the preparation method is shown as follows:

[0052]

[0053]

[0054] The present invention also provides a nanoparticle, which is prepared by adopting the above-mentioned bimetallic iridium complex conjugate, and the particle size of the nanoparticle is 100-200 nm.

[0055] The present invention also provides a method for producing nanoparticles, comprising:

[0056] The bimetallic iridium complex conjugate 4IrMn and distearoylphosphatidylethanolamine-polyethylene glycol are dissolved in a solvent at a ratio of (1-1.5) mg:1 mL, preferably tetrahydrofuran, and the dissolved solution is added dropwise into water. The mixture is stirred and evaporated at room temperature. The stirring and evaporation time is preferably 24 hours, and then dialyzed using a 2000 molecular weight dialysis membrane to obtain uniform and stable nanoparticles 4IrMn NPs.

[0057] The present invention also provides the use of the above-mentioned nanoparticles as a phototherapy material, and the phototherapy material is preferably a photosensitive initiator.

[0058] The present invention also provides use of the nanoparticles in preparing a drug for treating ischemic stroke.

[0059] Unless otherwise specified, the terms used herein have the meanings commonly understood by those skilled in the art. The technical solution of the present invention is described in more detail below with reference to the examples, in which the raw materials involved are all commercially available.

[0060] Example 1

[0061] The bimetallic iridium complex conjugate 4IrMn has the structural formula shown in Formula I:

[0062]

[0063] The preparation method of the above-mentioned bimetallic iridium complex conjugate is:

[0064] 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.

[0065] S2. To a 100 mL single-necked flask, add the phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2 (0.1 mmol, 0.1072 g) and porphyrin Schiff base (0.05 mmol, 0.0516 g) obtained above, followed by 60 mL of dichloromethane and methanol (v:v = 1:1) as the solvent. 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 to obtain a purple-red solid, namely 4Ir.

[0066] S3. 4Ir (0.1 mmol, 0.3033 g) and manganese chloride (0.5 mmol, 0.062 g) were added to a 100 mL beaker, and DMF was used as the reaction solvent. The reaction was stirred at 80°C for 9 h. The product was extracted with dichloromethane and water. The solvent in the system was removed by rotary evaporation and dried to obtain a purple-black solid product, namely the bimetallic iridium complex conjugate 4IrMn, with a yield of 80%. The molecular formula is C 156 H 108 Ir4MnN 20 , the relative molecular mass is 3087.69g / mol. Figure 1 The mass spectrometry data of the prepared 4IrMn showed a molecular weight of 3138.4 g / mol, which is consistent with the fitted molecular weight (4IrMn was sampled in acetonitrile, where the Mn element coordinated two acetonitrile molecules), proving the structure.

[0067] Example 2

[0068] 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.

[0069] S2. To a 100 mL single-necked flask, add the phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2 (0.1 mmol, 0.1072 g) and porphyrin Schiff base (0.05 mmol, 0.0516 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 extracted with dichloromethane and water. The resulting material was washed with petroleum ether and dried to obtain a purple-red solid, namely 4Ir.

[0070] S3. 4Ir (0.1 mmol, 0.3033 g) and manganese chloride (0.5 mmol, 0.062 g) were added to a 100 mL beaker, and DMF was used as the reaction solvent. The reaction was stirred at 80°C for 9 h. The product was extracted with dichloromethane and water. The solvent in the system was removed by rotary evaporation and dried to obtain a purple-black solid product, namely the bimetallic iridium complex conjugate 4IrMn, with a yield of 75%. The molecular formula is C 156 H 108 Ir4MnN 20 , the relative molecular mass is 3087.69g / mol.

[0071] Example 3

[0072] 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.

[0073] S2. To a 100 mL single-necked flask, add the phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2 (0.1 mmol, 0.1072 g) and porphyrin Schiff base (0.05 mmol, 0.0516 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 extracted with dichloromethane and water. The resulting material was washed with petroleum ether and dried to obtain a purple-red solid, namely 4Ir.

[0074] S3. 4Ir (0.1 mmol, 0.3033 g) and manganese chloride (0.5 mmol, 0.062 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 purple-black solid product, namely the bimetallic iridium complex conjugate 4IrMn, with a yield of 90%. The molecular formula is C 156 H 108 Ir4MnN 20 , the relative molecular mass is 3087.69g / mol.

[0075] Example 4

[0076] 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.

[0077] S2. To a 100 mL single-necked flask, add the above-obtained phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2 (0.1 mmol, 0.1072 g) and porphyrin Schiff base (0.05 mmol, 0.0516 g), followed by 60 mL of dichloromethane and methanol (v:v = 1:1) as the solvent. 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 4Ir as a purple-red solid.

[0078] S3. 4Ir (0.1 mmol, 0.3033 g) and manganese chloride (0.5 mmol, 0.062 g) were added to a 100 mL beaker, and DMF was used as the reaction solvent. The reaction was stirred at 80°C for 12 h. The product was extracted with dichloromethane and water. The solvent in the system was removed by rotary evaporation and dried to obtain a purple-black solid product, namely the bimetallic iridium complex conjugate 4IrMn, with a yield of 77% and a molecular formula of C 156 H 108 Ir4MnN 20 , the relative molecular mass is 3087.69g / mol.

[0079] Example 5

[0080] 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.

[0081] S2. To a 100 mL single-necked flask, add the phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2 (0.1 mmol, 0.1072 g) and porphyrin Schiff base (0.05 mmol, 0.0516 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 extracted with dichloromethane and water. The resulting material was washed with petroleum ether and dried to obtain a purple-red solid, namely 4Ir.

[0082] S3. 4Ir (0.1 mmol, 0.3033 g) and manganese chloride (0.5 mmol, 0.062 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 purple-black solid product, namely the bimetallic iridium complex conjugate 4IrMn, with a yield of 88%. The molecular formula is C 156 H 108 Ir4MnN 20 , the relative molecular mass is 3087.69g / mol.

[0083] Example 6

[0084] 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.

[0085] S2. To a 100 mL single-necked flask, add the phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2 (0.1 mmol, 0.1072 g) and porphyrin Schiff base (0.05 mmol, 0.0516 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 extracted with dichloromethane and water. The resulting material was washed with petroleum ether and dried to obtain a purple-red solid, namely 4Ir.

[0086] S3. 4Ir (0.1 mmol, 0.3033 g) and manganese chloride (0.5 mmol, 0.062 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 purple-black solid product, namely the bimetallic iridium complex conjugate 4IrMn, with a yield of 86%. The molecular formula is C 156 H 108 Ir4MnN 20 , the relative molecular mass is 3087.69g / mol.

[0087] The performance of the bimetallic iridium complex conjugate 4IrMn prepared in Example 1 was characterized as follows:

[0088] 1. Preparation of Nanoparticles

[0089] 4IrMn (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 the prepared 10 mL of water. The mixture was stirred and evaporated overnight at room temperature. After evaporation, 4IrMnNPs were dialyzed using a 2000 molecular weight dialysis membrane to obtain stable nanoparticles with uniform particle size.

[0090] 4Ir (0.0010 g) and distearoylphosphatidylethanolamine-polyethylene glycol (0.0020 g) prepared in step 2 of Example 1 were dissolved in 1 mL of tetrahydrofuran, respectively. The two were added dropwise to the prepared 10 mL of water, stirred and evaporated overnight at room temperature. After the end, dialysis was performed using a 2000 molecular weight dialysis membrane to obtain 4IrNPs nanoparticles with uniform particle size and stable dispersion in the aqueous solution.

[0091] IrMn (0.0010 g) 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 IrMn nanoparticles with uniform particle size and stable dispersion in the aqueous solution. The structural formula of IrMn is as follows:

[0092]

[0093] Ir (0.0010 g) 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 IrNPs with uniform particle size and stable dispersion in the aqueous solution. The structural formula of Ir is as follows:

[0094]

[0095] Tetraphenylporphyrin (TPP) (0.0010 g) and distearoylphosphatidylethanolamine-polyethylene glycol (0.0020 g) were dissolved in 1 mL of tetrahydrofuran, 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.

[0096] 2.4 Physical properties of IrMnNPs

[0097] Figure 2 is the particle size of TPP NPs, IrNPs, IrMnNPs, 4Ir, and 4IrMn NPs ( Figure 2 (a)) and potential diagram ( Figure 2 (b) (measured by a particle size potential analyzer). As can be seen in the figure, the particle size and potential of 4IrMn NPs in aqueous solution are 127 nm and -13.6 mV, respectively. The uniform particle size of <200 nm indicates good biocompatibility of 4IrMn NPs, and the negative potential suggests good circulation in the body.

[0098] 3.4 Photophysical properties of IrMnNPs

[0099] The UV absorption and fluorescence emission of 4IrMnNPs in aqueous solution were measured by UV-visible spectrophotometer and fluorescence spectrometer. Figure 3 As shown. Figure 3 (a) is the UV absorption spectrum, Figure 3 (b) is the fluorescence emission spectrum. The absorption in the ultraviolet absorption disappears because the Mn 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. When Mn is introduced, the fluorescence intensity is significantly reduced.

[0100] Figure 4 For the 4IrMnNPs prepared in the present invention, the singlet oxygen generation experiment and the related kinetic curves under the conditions of light and DPBF were measured by ultraviolet spectrophotometer, wherein Figure 4 (a) is the line graph of the DPBF decrease of NPs at a specific nanometer. Figure 4 (b) is the first-order kinetic curve corresponding to 4(a). The degradation of DPBF under light proves that CuIrNPs have good singlet oxygen production ability.

[0101] 4.4 Photothermal properties of IrMn NPs

[0102] Thermocouple photothermal data, where Figure 5 A is the temperature change diagram of different NPs, Figure 5 B is the temperature change diagram of different concentrations of NPs, where Figure 5 C is the temperature variation diagram of NPs with different powers, Figure 5 D is the five-time heating and cooling cycle diagram of 4IrMn NPs. Figure 5 Middle A shows that compared with the control group H2O, the drug groups (TPP NPs, Ir NPs, IrMn NPs, 4IrNPs, 4IrMn NPs) have better photothermal generation capabilities; Figure 5 It is shown in the figure that the higher the concentration of 4IrMn NPs, the higher the temperature under the same illumination time, indicating that it is concentration-dependent; Figure 5 The figure shows that 4IrMn NPs generate higher heat and higher temperature at different powers, indicating that it is power-dependent. Figure 5 The temperature changes of 4IrMnNP after five heating and cooling cycles are shown in Figure 2, which shows that the drug has good photothermal cycling ability.

[0103] Figure 6 This is the thermal imaging image of the photothermal data corresponding to 4IrMn NP. Under illumination conditions, the solution temperature increases with the extension of illumination time.

[0104] 5.4 Chemiluminescence experiment of IrMn NPs

[0105] Figure 7 The relevant data table of 4IrMn NP chemiluminescence was used to test the relevant chemiluminescence using IVIS mouse in vivo imaging system. Figure 7(a) Different types of RONS were co-incubated with 4IrMn NPs in a 96-well plate in response to different RONS. - The luminescence intensity was the strongest, and ONOO - As a marker substance, it can react with 4IrMnNP to produce chemiluminescence, illuminating the inflammatory site. Figure 7 (b), The chemiluminescence intensity of 4IrMn NPs is enhanced compared with the control group. Figure 7 (c) The dotted line graph of chemiluminescence intensity at different nanometers shows that the drug has the highest intensity of bioluminescence emission at 660 nm. Figure 7 (d), ONOO - The luminescence decay of 4IrMn NPs can reach 60min. Figure 7 (e) Using chicken breast of varying thicknesses to cover the drug, the chemiluminescence can penetrate up to 10 mm. This high penetration depth facilitates imaging of drugs at deep sites of inflammation, offering significant advantages over photosensitizers that rely on short-wavelength excitation light.

[0106] Figure 8 In vitro targeting testing of the 4IrMn 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.

[0107] 6.4 Antioxidant Activity Experiment of IrMnNPs

[0108] Figure 9 The test results of the antioxidant capacity of 4IrMnNPs are shown in Figure 2. Figure 9 ,in Figure 9 (a) is the DPPH degradation diagram, Figure 9 (b) is a graph of hydrogen peroxide degradation. Detection using a kit shows that when the concentration of 4IrMnNPs reaches 300 μg / mL, the consumption rate of DPPH can reach about 82%. Figure 9 The consumption rate of 4IrMnNPs was detected to be nearly 70% after co-incubation with hydrogen peroxide, which proved that 4IrMnNPs had good antioxidant capacity and showed that they had a good therapeutic effect on inflammation in vivo.

[0109] 7.4 In vitro thrombolysis experiments of IrMnNPs

[0110] 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 10 As shown. Figure 10 (a) is a diagram showing the rapid changes in thrombus size before and after thrombolysis, where (1 represents PBS, 2 represents UK, 3 represents TPP NPs, 4 represents IrNPs, 5 represents IrMn NPs; 6 represents 4Ir NPs; 7 represents 4IrMn NPs; the corresponding figure below is the corresponding light exposure group). Figure 10 (b) shows the thrombus dissolution rate. Compared with the control group, the 4IrMnNPs group can simultaneously activate PDT / PTT dual-mode thrombolysis under laser irradiation, with a thrombolysis rate of 58%. The experimental results show that 4IrMnNPs have excellent in vitro thrombolytic ability.

[0111] 8.4 Intracellular Antioxidant Experiment of IrMnNPs

[0112] Figure 11 4IrMn NPs were co-incubated with HT22 cells that produced a large amount of RONS induced by H2O2, and the scavenging of total ROS, superoxide anions and hydroxyl radicals by 4IrMnNPs were monitored using DCFH-DA, DHE and HPF probes, respectively, demonstrating that 4IrMnNPs have good antioxidant capacity.

[0113] 9.4 In vivo experiments with IrMnNPs in mice

[0114] Figure 12 In vivo chemiluminescence imaging of 4IrMnNPs. Following tail vein administration, the drug circulates to the thrombus over time, with pronounced chemiluminescence observed under illumination. Maximum aggregation is achieved around 60 minutes, signaling the start of thrombolytic therapy.

[0115] Figure 13 Figure 4 shows an in vivo thrombolytic test of 4IrMnNPs. Anesthetized mice were thoracotomized to locate the right carotid artery. The artery was then incubated with 10% FeCl3 solution for approximately 10 minutes. The vessel turned black, and the thrombus formation was complete. After drug administration, the drug circulated to the thrombus site. Laser (635nm 0.8w / cm 2 ) irradiates the thrombus location, and the thrombolysis rate in the body reaches 70%, which has a good thrombolytic effect.

[0116] Figure 14 The chemiluminescence of 4IrMn NPs was monitored by a small animal imaging instrument within 24 hours after brain administration. Figure 14 (a) Chemiluminescence imaging of 4IrMn NPs at the stroke site at 0, 3, 6, 9, 12 and 24 hours. Figure 14 (b) corresponds to Figure 14 (a) Quantification of the luminescence level, Figure 14 It is shown that the present invention realizes the visual monitoring of diseases.

[0117] Figure 15 TTC staining of the mouse brains after administration of 4IrMn NPs, sham operation group and PBS group showed that the white infarct area was significantly reduced in the 4IrMn NPs group, indicating that 4IrMn NPs have a significant effect on rescuing brain damage.

[0118] Figure 16 The movement trajectories of mice were monitored after administration of 4IrMn NPs, sham operation group and PBS group. The activity trajectories of mice increased significantly after administration of 4IrMn NPs, indicating that 4IrMn NPs played a positive role in the recovery of motor function of mice.

[0119] 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 of the bimetallic iridium complex conjugate is shown in formula (I): （Ⅰ）。 2. The method for preparing a bimetallic iridium complex conjugate according to claim 1, wherein: The following steps are involved: S1. Under nitrogen protection, IrCl3·3H2O and phenylpyridine ligand were heated to reflux to react to obtain phenylpyridine iridium dichloro bridge [Ir(ppy)2Cl2]2; S2. Under nitrogen, the phenylpyridine iridium dichloro bridge and porphyrin Schiff base ligand prepared in S1 were placed in the dark under the action of a solvent and refluxed. After the reaction, potassium hexafluorophosphate was added and stirred to obtain a tetranuclear iridium complex 4Ir. S3. Under nitrogen protection, the tetranuclear iridium complex 4Ir prepared in S2 is mixed with manganese chloride to react to obtain a bimetallic iridium complex conjugate 4IrMn.

3. 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 to the porphyrin Schiff base ligand is 1:

1.

4. The method for preparing a bimetallic iridium complex conjugate according to claim 2, wherein: In step S2, the reflux reaction temperature is 75-80° C., and the reflux reaction time is 8 h.

5. The method for preparing a bimetallic iridium complex conjugate according to claim 2, wherein: In step S3, the molar ratio of the tetranuclear metal iridium complex 4Ir to manganese chloride is 1:(5-7).

6. The method for preparing a bimetallic iridium complex conjugate according to claim 2, wherein: In step S3, the reaction temperature is 75° C.-80° C., and the reaction time is 9-12 h.

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 100-200 nm.

8. The method of claim 7, wherein: include: The bimetallic iridium complex conjugate 4IrMn and distearoylphosphatidylethanolamine-polyethylene glycol were dissolved in solvents respectively, and the dissolved solutions were added dropwise into water. The mixture was stirred and evaporated at room temperature, and then dialyzed to obtain nanoparticles 4IrMn NPs.

9. Use of the nanoparticles obtained by the preparation method of claim 7 or claim 8 in the preparation of phototherapy materials.

10. Use of the nanoparticles obtained by the preparation method of claim 7 or claim 8 in preparing a drug for treating ischemic stroke.

Images