Bimetallic iridium complex conjugate, nanoparticle, preparation method and application in ischemic stroke

Abstract

The invention provides a bimetallic iridium complex conjugate, nanoparticles, a preparation method and application in ischemic stroke, and belongs to the technical field of photodynamic therapy drugs. The structural formula of the bimetallic iridium complex conjugate is as shown in formula (I). The invention also provides a preparation method of the bimetallic iridium complex conjugate. The invention further provides nanoparticles, the prepared bimetallic iridium complex has the chemiluminescence property, the photothermal and photodynamic synergistic thrombolysis treatment effect can be achieved at the same time, and the oxidation resistance of the bimetallic iridium complex has the remarkable treatment effect on inhibiting nerve cell death and rescuing infarction brain injury parts. The conjugate provided by the invention has good photodynamic photo-thermal capability, and can overcome the problems of short excitation wavelength of exciting light and limited light penetration depth required by the existing metal iridium complex. Experiments show that the material has a good treatment 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, real-time controllable light stimulation method with broad application prospects in the field of disease treatment. Compared with traditional drugs and surgery, phototherapy has inherent advantages such as non-invasiveness, low toxicity, simple operation, and fast recovery, and has attracted much attention in the medical field.

[0003] Surgical removal of emboli or drug thrombolysis of ischemic stroke is the main method for clinical treatment of thrombotic diseases. However, current thrombolytic drugs often show only limited therapeutic effects due to their low selectivity, short half-life and non-negligible risk of bleeding. In recent years, the application of photosensitizers in the diagnosis and treatment of cardiovascular and cerebrovascular diseases in a non-invasive manner has attracted widespread attention, especially in the field of thrombolysis, and has the potential to become a new generation of treatment methods.

[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 essential 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 will inevitably damage 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 infarct area, which releases 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 - level, thus facilitating early diagnosis of ischemic stroke. This approach is of great benefit in alleviating the adverse effects of neuronal death on brain function (such as limited motor function and impaired consciousness). Therefore, ischemic stroke urgently needs a comprehensive treatment method that can achieve safe thrombolysis, monitor and remove RONS, and save the ischemic penumbra near the infarct core, thereby minimizing the final infarct area and improving neurological deficits.

[0005] Although PTT has shown some potential in thrombolytic therapy, the nonspecific thermal damage of PTT 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 can be used in combination with other therapies. However, the clinical application of PDT is also limited by its dependence on oxygen, insufficient ROS generation efficiency of photosensitizers, and like PTT, there is a problem of limited penetration depth due to insufficient wavelength of the light source.

[0006] As research deepens, the integration of PTT and PDT into a single system for synergistic treatment shows a promising prospect of high efficiency and safety. The oxygen independence of PTT can make up for the oxygen dependence of PDT. At the same time, the local thermal effect of PTT can accelerate local blood circulation and increase local oxygen supply, promoting local PDT.

[0007] Transition metal materials are favored for their rich excited state properties, such as high luminescence quantum efficiency, ultra-long luminescence lifetime, high stability luminescence, 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, and are currently the most widely used type 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, and used the synergistic effect of photodynamics and photothermal to achieve efficient anti-thrombotic therapeutic effects. However, the synthesis of this photosensitizer is relatively complex, which limits its wide 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 light 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 the property of chemiluminescence, and can realize visual monitoring in the early stage of disease development. At the same time, its excellent antioxidant property has excellent therapeutic effects on the rescue of ischemic penumbra and the reduction of cerebral infarction area.

[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 to reflux 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 a dark place under the action of a solvent for reflux reaction. After the reaction, potassium hexafluorophosphate was added and stirred continuously to obtain a tetranuclear metal iridium complex 4Ir;

[0015] S3. Under nitrogen protection, the tetranuclear metal iridium complex 4Ir prepared in S2 is mixed with manganese chloride to react 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 time of the reflux reaction 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 9-12h.

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

[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, stirred and evaporated at room temperature, and dialyzed to obtain nanoparticles 4IrMnNPs.

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

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

[0025] Beneficial Effects of the Invention

[0026] The present invention provides a bimetallic iridium complex conjugate, nanoparticles, a preparation method and 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 the property of chemiluminescence, and can realize visual monitoring in the early stage of disease development. At the same time, its excellent antioxidant property has excellent therapeutic effects on the rescue of ischemic penumbra and the reduction of cerebral infarction area.

[0027] The present invention also provides nanoparticles 4IrMn NPs formed by biologically encapsulating the bimetallic iridium complex conjugate 4IrMn into the 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 the present invention can react chemically with excessive ROS at the inflammatory site without the need for an external excitation light source to produce bioluminescence. Experiments show that it has a high penetration of 10 mm, which can overcome the problem of short excitation wavelength and limited light penetration depth required by the existing metal iridium complexes. Test results show that CuIr NPs have good antioxidant capacity, which indicates that the material has a good therapeutic effect on stroke and is expected to be developed as a phototherapy material and used 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, in which the particle sizes and potentials of TPP NPs, IrNPs, IrMn NPs, 4IrNPs and 4IrMn NPs are respectively corresponded.

[0031] Figure 3Ultraviolet 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), the larger the slope, the more singlet oxygen is produced.

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

[0034] Figure 6 This is a thermal imaging data diagram of 4IrMnNPs prepared in Example 1 of the present invention in an 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 selectivity intensity graphs 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] Fig. 9 This is a graph showing the antioxidant capacity test of 4IrMn NPs prepared in Example 1 of the present invention, including a total antioxidant capacity test and hydrogen peroxide consumption.

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

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

[0040] Fig.12 This is a test diagram of the chemiluminescence monitoring of mouse thrombosis using 4IrMnNPs prepared in Example 1 of the present invention.

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

[0042] Fig.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] Fig.15 This is a TTC staining image of brain slices of mice treated with 4IrMn NPs in Example 1 of the present invention.

[0044] Fig.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. Add IrCl3·3H2O and phenylpyridine ligand to a reaction vessel containing solvent and water, and heat and reflux under nitrogen protection, preferably reflux at 120-130°C for 24-30h, wait for the reaction to cool to room temperature, add a large amount of poor solvent water to precipitate and filter, rinse the solvent with a large amount of water and ethanol, and dry the obtained solid 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 container, then add a solvent, the solvent is preferably a mixture of dichloromethane and methanol (v:v=1:1), and place the reaction in a dark place under the condition of fully filling with inert gas N2 for reflux reaction. The reflux reaction temperature is preferably 75-80°C, and the reaction time is preferably 8h. When the reaction is completed and cooled to room temperature, add potassium hexafluorophosphate solid to the solution in the bottle, and continue to react at room temperature. The method comprises the steps of: stirring for a preferred stirring time of 45 to 60 minutes, removing the solvent in the system by a rotary evaporator, extracting with dichloromethane and water to remove excess potassium hexafluorophosphate solid, washing the obtained substance with petroleum ether and drying it, and purifying it by column chromatography to obtain a purple-red solid, namely, a 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; and 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, and 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, i.e., 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 using the above-mentioned bimetallic iridium complex conjugate, and the particle size of the nanoparticle is 100-200nm.

[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, and the solvent is preferably tetrahydrofuran. The dissolved solutions are added dropwise into water, stirred and evaporated at room temperature, and the stirring and evaporation time is preferably 24 hours. 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 the 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 scheme of the present invention is described in more detail below in conjunction with the examples, and the raw materials involved in the examples are all commercially available.

[0060] Example 1

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

[0062]

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

[0064] S1. Add IrCl3·3H2O (0.1mmol, 0.0352g) and phenylpyridine ligand (0.3mmol, 0.0471g) to a round-bottom flask containing 30mL of 2-ethylene glycol ether and 10mL of water. Heat and reflux at 120°C for 24h under N2 protection. After the reaction is cooled to room temperature, add a large amount of poor solvent water to precipitate the precipitate, filter it, and wash it several times with a large amount of water and ethanol solvent. The solid obtained by drying is phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2.

[0065] S2. Add the above-obtained phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2 (0.1mmol, 0.1072g) and porphyrin Schiff base (0.05mmol, 0.0516g) to a 100mL single-mouth bottle, and then add 60mL of dichloromethane and methanol (v:v=1:1) as the solvent of the reaction system. With the inert gas N2 fully filled, place the reaction in a dark place for reflux reaction, reflux reaction at 80°C for 8h, and when the reaction is completed and cooled to room temperature, add 10 equivalents of potassium hexafluorophosphate solid to the solution in the bottle, continue stirring at room temperature for 45min, remove the solvent in the system by rotary evaporator, extract with dichloromethane and water to remove excess potassium hexafluorophosphate solid, and wash the obtained substance with petroleum ether and dry it to obtain a purple-red solid, namely 4Ir.

[0066] S3. Add 4Ir (0.1mmol, 0.3033g) and manganese chloride (0.5mmol, 0.062g) into a 100mL beaker, use DMF as the reaction solvent, stir and react at 80℃ for 9h, extract the product with dichloromethane and water, remove the solvent in the system by rotary evaporator, and dry to obtain a purple-black solid product, i.e., bimetallic iridium complex conjugate 4IrMn, with a yield of 80%, and a molecular formula of C 156 H 108 Ir4Mn 20 , the relative molecular mass is 3087.69g / mol. Figure 1 The mass spectrum data of the prepared 4IrMn shows a molecular weight of 3138.4 g / mol, which is the same as the fitted molecular weight (4IrMn is sampled in acetonitrile, where the Mn element coordinates two acetonitrile molecules), which proves the structure.

[0067] Example 2

[0068] S1. Add IrCl3·3H2O (0.1mmol, 0.0352g) and phenylpyridine ligand (0.25mmol, 0.0392g) to a round-bottom flask containing 30mL of 2-ethylene glycol ether and 10mL of water. Heat and reflux at 120°C for 24h under N2 protection. After the reaction is cooled to room temperature, add a large amount of poor solvent water to precipitate the precipitate, filter it, and wash it several times with a large amount of water and ethanol solvent. The solid obtained by drying is phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2.

[0069] S2. Add the above-obtained phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2 (0.1mmol, 0.1072g) and porphyrin Schiff base (0.05mmol, 0.0516g) to a 100mL single-mouth bottle, and then add 60mL of dichloromethane and methanol (v:v=1:1) as the solvent of the reaction system. With the inert gas N2 fully filled, place the reaction in a dark place for reflux reaction, reflux reaction at 80°C for 8h, and when the reaction is completed and cooled to room temperature, add 10 equivalents of potassium hexafluorophosphate solid to the solution in the bottle, continue stirring at room temperature for 45min, remove the solvent in the system by rotary evaporator, extract with dichloromethane and water to remove excess potassium hexafluorophosphate solid, and wash the obtained substance with petroleum ether and dry to obtain a purple-red solid, namely 4Ir.

[0070] S3. Add 4Ir (0.1mmol, 0.3033g) and manganese chloride (0.5mmol, 0.062g) into a 100mL beaker, use DMF as the reaction solvent, stir and react at 80℃ for 9h, extract the product with dichloromethane and water, remove the solvent in the system by rotary evaporator, and dry to obtain a purple-black solid product, i.e., bimetallic iridium complex conjugate 4IrMn, with a yield of 75%, and the molecular formula is C 156 H 108 Ir4Mn 20 , the relative molecular mass is 3087.69g / mol.

[0071] Example 3

[0072] S1. Add IrCl3·3H2O (0.1mmol, 0.0352g) and phenylpyridine ligand (0.3mmol, 0.0471g) to a round-bottom flask containing 30mL of 2-ethylene glycol ether and 10mL of water. Heat and reflux at 120°C for 30h under N2 protection. After the reaction is cooled to room temperature, add a large amount of poor solvent water to precipitate the precipitate, filter it, and wash it several times with a large amount of water and ethanol solvent. The solid obtained by drying is phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2.

[0073] S2. Add the above-obtained phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2 (0.1mmol, 0.1072g) and porphyrin Schiff base (0.05mmol, 0.0516g) to a 100mL single-mouth bottle, and then add 60mL of dichloromethane and methanol (v:v=1:1) as the solvent of the reaction system. With the inert gas N2 fully filled, place the reaction in a dark place for reflux reaction, reflux reaction at 75°C for 8h, and when the reaction is completed and cooled to room temperature, add 10 equivalents of potassium hexafluorophosphate solid to the solution in the bottle, continue stirring at room temperature for 45min, remove the solvent in the system by rotary evaporator, extract with dichloromethane and water to remove excess potassium hexafluorophosphate solid, and wash the obtained substance with petroleum ether and dry to obtain a purple-red solid, namely 4Ir.

[0074] S3. Add 4Ir (0.1mmol, 0.3033g) and manganese chloride (0.5mmol, 0.062g) into a 100mL beaker, use DMF as the reaction solvent, stir and react at 80℃ for 8h, extract the product with dichloromethane and water, remove the solvent in the system by rotary evaporator, and dry to obtain a purple-black solid product, i.e., bimetallic iridium complex conjugate 4IrMn, with a yield of 90%, and the molecular formula is C 156 H 108 Ir4Mn 20 , the relative molecular mass is 3087.69g / mol.

[0075] Example 4

[0076] S1. Add IrCl3·3H2O (0.1mmol, 0.0352g) and phenylpyridine ligand (0.3mmol, 0.0471g) to a round-bottom flask containing 30mL of 2-ethylene glycol ether and 10mL of water. Heat and reflux at 130°C for 24h under N2 protection. After the reaction is cooled to room temperature, add a large amount of poor solvent water to precipitate the precipitate, filter it, and wash it several times with a large amount of water and ethanol solvent. The solid obtained by drying is phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2.

[0077] S2. Add the above-obtained phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2 (0.1mmol, 0.1072g) and porphyrin Schiff base (0.05mmol, 0.0516g) to a 100mL single-mouth bottle, and then add 60mL of dichloromethane and methanol (v:v=1:1) as the solvent of the reaction system. When the inert gas N2 is fully filled, the reaction is placed in a dark place for reflux reaction, and the reflux reaction is carried out at 75°C for 8h. When the reaction is completed and cooled to room temperature, 10 equivalents of potassium hexafluorophosphate solid are added to the solution in the bottle, and stirring is continued at room temperature for 45min. The solvent in the system is removed by a rotary evaporator, and the excess potassium hexafluorophosphate solid is removed by extraction with dichloromethane and water, and the obtained substance is washed with petroleum ether and dried, and purified by column chromatography to obtain a purple-red solid, namely 4Ir.

[0078] S3. Add 4Ir (0.1mmol, 0.3033g) and manganese chloride (0.5mmol, 0.062g) into a 100mL beaker, use DMF as the reaction solvent, stir and react at 80℃ for 12h, extract the product with dichloromethane and water, remove the solvent in the system by rotary evaporator, and dry to obtain a purple-black solid product, i.e., bimetallic iridium complex conjugate 4IrMn, with a yield of 77% and a molecular formula of C 156 H 108 Ir4Mn 20 , the relative molecular mass is 3087.69g / mol.

[0079] Example 5

[0080] S1. Add IrCl3·3H2O (0.1mmol, 0.0352g) and phenylpyridine ligand (0.3mmol, 0.0471g) to a round-bottom flask containing 30mL of 2-ethylene glycol ether and 10mL of water. Heat and reflux at 120°C for 24h under N2 protection. After the reaction is cooled to room temperature, add a large amount of poor solvent water to precipitate the precipitate, filter it, and wash it several times with a large amount of water and ethanol solvent. The solid obtained by drying is phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2.

[0081] S2. Add the above-obtained phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2 (0.1mmol, 0.1072g) and porphyrin Schiff base (0.05mmol, 0.0516g) to a 100mL single-mouth bottle, and then add 60mL of dichloromethane and methanol (v:v=1:1) as the solvent of the reaction system. With the inert gas N2 fully filled, place the reaction in a dark place for reflux reaction, reflux reaction at 80°C for 8h, and when the reaction is completed and cooled to room temperature, add 10 equivalents of potassium hexafluorophosphate solid to the solution in the bottle, continue stirring at room temperature for 45min, remove the solvent in the system by rotary evaporator, extract with dichloromethane and water to remove excess potassium hexafluorophosphate solid, and wash the obtained substance with petroleum ether and dry to obtain a purple-red solid, namely 4Ir.

[0082] S3. Add 4Ir (0.1mmol, 0.3033g) and manganese chloride (0.5mmol, 0.062g) into a 100mL beaker, use DMF as the reaction solvent, stir and react at 80℃ for 8h, extract the product with dichloromethane and water, remove the solvent in the system by rotary evaporator, and dry to obtain a purple-black solid product, i.e., bimetallic iridium complex conjugate 4IrMn, with a yield of 88% and a molecular formula of C 156 H 108 Ir4Mn 20 , the relative molecular mass is 3087.69g / mol.

[0083] Example 6

[0084] S1. Add IrCl3·3H2O (0.1mmol, 0.0352g) and phenylpyridine ligand (0.3mmol, 0.0471g) to a round-bottom flask containing 30mL of 2-ethylene glycol ether and 10mL of water. Heat and reflux at 120°C for 24h under N2 protection. After the reaction is cooled to room temperature, add a large amount of poor solvent water to precipitate the precipitate, filter it, and wash it several times with a large amount of water and ethanol solvent. The solid obtained by drying is phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2.

[0085] S2. Add the above-obtained phenylpyridine iridium dichlorobridge [Ir(ppy)2Cl2]2 (0.1mmol, 0.1072g) and porphyrin Schiff base (0.05mmol, 0.0516g) to a 100mL single-mouth bottle, and then add 60mL of dichloromethane and methanol (v:v=1:1) as the solvent of the reaction system. With the inert gas N2 fully filled, place the reaction in a dark place for reflux reaction, reflux reaction at 80°C for 8h, and when the reaction is completed and cooled to room temperature, add 10 equivalents of potassium hexafluorophosphate solid to the solution in the bottle, continue stirring at room temperature for 45min, remove the solvent in the system by rotary evaporator, extract with dichloromethane and water to remove excess potassium hexafluorophosphate solid, and wash the obtained substance with petroleum ether and dry to obtain a purple-red solid, namely 4Ir.

[0086] S3. Add 4Ir (0.1mmol, 0.3033g) and manganese chloride (0.5mmol, 0.062g) into a 100mL beaker, use DMF as the reaction solvent, stir and react at 77°C for 8h, extract the product with dichloromethane and water, remove the solvent in the system by rotary evaporator, and dry to obtain a purple-black solid product, i.e., bimetallic iridium complex conjugate 4IrMn, with a yield of 86% and a molecular formula of C 156 H 108 Ir4Mn 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, respectively, and then added dropwise to the prepared 10 mL of water. The mixture was stirred and evaporated overnight at room temperature. After the mixture was evaporated, it was dialyzed using a 2000 molecular weight dialysis membrane to obtain stable nanoparticles 4IrMnNPs with uniform particle size.

[0090] The 4Ir (0.0010 g) prepared in step 2 of Example 1 and distearoyl phosphatidylethanolamine-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, a 2000 molecular weight dialysis membrane was used for dialysis to obtain 4IrNPs nanoparticles with uniform particle size and stable dispersion in 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 the prepared 10 mL of water, stirred and evaporated overnight at room temperature. After the evaporation, 2000 molecular weight dialysis membrane was used for dialysis to obtain nanoparticles IrMnNPs with uniform particle size and stable dispersion in aqueous solution. The structural formula of IrMn is as follows:

[0092]

[0093] Ir (0.0010 g) and distearoyl phosphatidylethanolamine-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 the mixture was dialyzed using a 2000 molecular weight dialysis membrane, IrNPs with uniform particle size and stable dispersion in the aqueous solution were obtained. 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, respectively, and then added dropwise to the prepared 10 mL of water. The mixture was stirred and evaporated overnight at room temperature. After the mixture was evaporated, it was dialyzed using a 1000 molecular weight dialysis membrane to obtain TPPNPs nanoparticles with uniform particle size and stable dispersion in 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 particle size potential analyzer), it can be seen from the figure that the particle size and potential of 4IrMn NPs in aqueous solution are 127nm and -13.6mV respectively. The uniform size of the particle size <200nm indicates that 4IrMn NPs have good biocompatibility and the potential is negative, indicating that it can have a good circulation effect 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 two original Q bands of porphyrin disappear in the ultraviolet absorption because Mn metal enters the porphyrin core. The emission intensity becomes weaker due to the introduction of Ir core. After the introduction of Mn, the fluorescence intensity decreases significantly.

[0100] Figure 4 For the 4IrMnNPs prepared in the present invention, the singlet oxygen generation experiment in vitro under the conditions of light and DPBF and the related kinetic curves were measured by ultraviolet spectrophotometer, wherein Figure 4 (a) is the line graph of 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 NPs with different concentrations. Figure 5 C is the temperature variation diagram of NPs with different powers, Figure 5 D is the five heating and cooling cycles 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 It is shown in the figure that the heat generation and temperature rise of 4IrMn NPs at different powers are higher with higher power, 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 of 4IrMn NP. Under illumination conditions, the solution temperature increases with the extension of illumination time.

[0104] 5.4 Chemiluminescence experiment of IrMn NP

[0105] Figure 7 The relevant data table of 4IrMn NP chemiluminescence was used to test the relevant chemiluminescence using IVIS mouse in vivo imaging instrument. 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. - As a marker substance, it can react with 4IrMnNP to produce chemiluminescence, illuminating the inflammatory site. Figure 7 (b), compared with the control group, the chemiluminescence intensity of 4IrMn NPs is enhanced. 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 660nm. Figure 7 (d), ONOO - The luminescence decay of 4IrMn NPs can reach 60min. Figure 7 (e) Using chicken breasts of different thicknesses to cover the drug, the chemiluminescence can penetrate up to 10 mm. The high penetration depth is conducive to imaging tests of drugs at deep inflammatory sites. This has significant advantages over photosensitizers that rely on short-wavelength excitation light.

[0106] Figure 8 This is an in vitro targeting test of the 4IrMn NPs of the present invention. Fresh mouse blood was added to a 5 mL test tube without anticoagulant and refrigerated at 4°C for 24 hours to prepare a thrombus. The drug was co-incubated with the thrombus, and the chemiluminescence intensity was detected at 2 hours, 4 hours, and 6 hours. The results showed that the longer the co-incubation time with the drug, the higher the chemiluminescence intensity, which proved that the drug had a good targeting ability for the thrombus.

[0107] 6.4 Antioxidant properties of IrMnNPs

[0108] Fig. 9 The test results of the antioxidant capacity of 4IrMnNPs. Fig. 9 ,in Fig. 9 (a) is the DPPH degradation diagram, Fig. 9 (b) is a hydrogen peroxide degradation diagram. The test kit was used to detect that when the concentration of 4IrMnNPs reached 300 μg / mL, the consumption rate of DPPH could reach about 82%. Fig. 9 , and its consumption rate can be detected by co-incubation with hydrogen peroxide, which is nearly 70%. This proves that 4IrMnNPs have good antioxidant capacity and show that it has a good therapeutic effect on inflammation in vivo.

[0109] 7.4 In vitro thrombolysis experiment of IrMnNPs

[0110] 5 mL of NPs from different groups and thrombus were placed in 10 mL vials for in vitro thrombolysis test. Fig.10 As shown. Fig.10 (a) is a diagram showing the rapid changes of 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 illumination group). Fig.10 (b) is a graph showing the thrombus dissolution rate. Compared with the control group, the 4IrMnNPs group can simultaneously activate PDT / PTT dual-mode thrombolysis under laser irradiation, and the thrombolysis rate reached 58%. The experimental results show that 4IrMnNPs has a good in vitro thrombolytic ability.

[0111] 8.4 Intracellular antioxidant experiment of IrMnNPs

[0112] Fig.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] Fig.12 This is the in vivo chemiluminescence imaging of 4IrMnNPs. It can be seen that after the tail vein administration, as time goes by, the drug circulates to the location of the thrombus, the chemiluminescence phenomenon is obvious under light, and the aggregation degree reaches the maximum at 60 minutes, and thrombolytic therapy can be started.

[0115] Fig.13 This is a test image of 4IrMnNPs in vivo thrombolysis. The mouse was anesthetized and the right carotid artery was found. The 10% FeCl3 solution was incubated for about 10 minutes. The blood vessels turned black and the thrombus formation was completed. After the drug was administered, it circulated to the thrombus position. The 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] Fig.14 The chemiluminescence of 4IrMn NPs was monitored by a small animal imaging instrument within 24 hours after brain administration. Fig.14 (a) Chemiluminescence imaging of 4IrMn NPs at the stroke site at 0, 3, 6, 9, 12 and 24 h. Fig.14 (b) corresponds to Fig.14 (a) Quantification diagram of the luminescence level, Fig.14 It is shown that the present invention realizes the visual monitoring of diseases.

[0117] Fig.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] Fig.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 4IrMnNPs, which indicates that 4IrMn NPs played a positive role in the recovery of motor function of mice.

[0119] The above implementation modes are only used to illustrate the technical solutions of the present invention, but not to limit the present invention. Although the present invention has been described in detail with reference to the above implementation modes, 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 protection scope 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):

2. The method for preparing the 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 to reflux to obtain phenylpyridine iridium dichloro bridge [Ir(ppy)2Cl2]2; S2. Under nitrogen protection, the phenylpyridine iridium dichloro bridge and porphyrin Schiff base ligand prepared in S1 were placed in a dark place under the action of a solvent for reflux reaction. After the reaction, potassium hexafluorophosphate was added and stirred continuously to obtain a tetranuclear metal iridium complex 4Ir; S3. Under nitrogen protection, the tetranuclear metal 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 the bimetallic iridium complex conjugate according to claim 2, characterized in that: In step S2, the molar ratio of the phenylpyridine iridium dichloro bridge and the porphyrin Schiff base ligand is 1:

1.

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

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

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

8. The method of a nanoparticle according to claim 7, characterized in that: 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, stirred and evaporated at room temperature, and dialyzed to obtain nanoparticles 4IrMn NPs.

9. Use of the nanoparticles obtained by the preparation method of claim 7 or claim 8 as 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