Multifunctional iridium (III) complex and preparation method and application thereof

Abstract

The present application belongs to the technical field of coordination chemistry and biomedical science, and provides a multifunctional iridium (III) complex, a preparation method and application thereof. The multifunctional iridium (III) complex (Mito-Ir) is composed of an iridium (III) complex cation and a coordination anion shown in the following formula. Mito-Ir can efficiently target mitochondria, and also has the abilities of phosphorescence imaging, type I and type II active oxygen generation, and photocatalytic oxidation of nicotinamide adenine dinucleotide. Under light irradiation, Mito-Ir triggers severe mitochondrial dysfunction through the above synergistic effect, and then specifically activates the caspase-3 / GSDME signaling pathway, and significantly induces pyroptosis; this pyroptosis-based cell death mechanism can effectively overcome the apoptosis tolerance of tumor cells, and is accompanied by the release of a large amount of inflammatory factors and damage-associated molecular patterns, stimulates immunogenic cell death, and activates the body's anti-tumor immune response.

Description

Technical Field

[0001] This invention relates to the interdisciplinary fields of coordination chemistry and biomedicine, and in particular to a multifunctional iridium(III) complex, its preparation method, and its applications. Background Technology

[0002] Photodynamic therapy (PDT), as a non-invasive treatment with high spatiotemporal selectivity, has shown great potential in cancer treatment. Its core mechanism relies on photosensitizers generating reactive oxygen species (ROS) under specific light irradiation, thereby inducing tumor cell death. However, traditional photosensitizers, especially porphyrin-based formulations, face several key technical bottlenecks in practical applications: First, the therapeutic effect is severely limited by the tumor microenvironment (TME). Most traditional photosensitizers follow a single type II photodynamic mechanism, and their killing effect is highly dependent on oxygen molecules to generate singlet oxygen. However, solid tumors are typically in a highly hypoxic state, which directly leads to insufficient ROS production, significantly weakening the therapeutic effect of PDT and even inducing treatment tolerance.

[0003] Secondly, the cell death mechanism is singular and prone to tolerance. Currently, PDT-induced cell death mainly relies on apoptosis. However, many malignant tumor cells acquire significant apoptosis resistance through mechanisms such as upregulation of anti-apoptotic proteins (e.g., Bcl-2). This makes apoptosis-dependent therapies less effective against such drug-resistant tumors, posing a major challenge in clinical treatment.

[0004] Furthermore, existing photosensitizers have limited functionality, making it difficult to achieve integrated diagnosis and treatment and synergistic effects. Although iridium(III) complexes with excellent optical properties and tunability have attracted much attention as a new generation of photosensitizers in recent years, their functions are often limited to ROS generation or single organelle targeting. Developing "integrated" smart photosensitizers that can simultaneously integrate multiple functions (such as targeting, imaging, multimodal ROS generation, and bioenergy intervention) remains a major challenge in this field.

[0005] Finally, its ability to actively induce immunogenic cell death (ICD) is insufficient. An ideal antitumor therapy should not only directly kill tumor cells but also activate the body's own antitumor immunity, producing a systemic and long-lasting "distant effect." However, the traditional apoptosis process is generally considered immune-silencing or inhibitory, making it difficult to effectively elicit a strong immune response.

[0006] In recent years, an inflammatory programmed cell death mechanism known as pyroptosis has offered a novel approach to overcoming the aforementioned challenges. Pyroptosis is characterized by cell swelling, membrane pore formation, and the release of large amounts of pro-inflammatory factors and damage-associated molecular patterns (DAMPs), which can efficiently induce intracellular cytotoxicity (ICD) and activate T cell-mediated anti-tumor immunity. Therefore, developing novel photosensitizers that can directly and efficiently induce pyroptosis in tumor cells holds promise for simultaneously addressing the two major challenges of "apoptosis resistance" and "insufficient immune activation," representing a highly promising direction in the field of phototherapy (PDT).

[0007] In summary, there is an urgent need in this field for a novel photosensitizer that can: (1) overcome the limitations of the hypoxic microenvironment of tumors; (2) circumvent the apoptosis resistance pathway of tumor cells; (3) possess multiple synergistic functions to enhance therapeutic efficacy; and (4) effectively induce immunogenic pyroptosis and activate anti-tumor immunity. Summary of the Invention

[0008] In view of this, the purpose of this invention is to provide a multifunctional iridium(III) complex, its preparation method, and its applications. The multifunctional iridium(III) complex of this invention targets mitochondria, synergistically generates type I / II ROS, photocatalytically oxidizes NADH, and / or induces pyroptosis and further triggers immunogenic cell death by activating the caspase-3 / GSDME signaling pathway.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a multifunctional iridium(III) complex, which is composed of an iridium(III) complex cation unit with the structure shown in Formula I and a corresponding coordinating anion: Formula I.

[0010] Preferably, the coordinating anion includes [PF6]. - [BF4] - Or [ClO4] - .

[0011] The present invention also provides a method for preparing the multifunctional iridium(III) complex described in the above technical solution, comprising the following steps: Ir2(ppy)4Cl2 having the structure shown in Formula a and 3,8-bis(thiophene-2-yl)-1,10-phenanthroline were dissolved in an organic solvent and subjected to a coordination reaction. An ion exchange reagent containing anion was added to the obtained coordination reaction solution to carry out ion exchange, thereby obtaining the multifunctional iridium(III) complex. Formula a.

[0012] Preferably, the molar ratio of Ir2(ppy)4Cl2 to 3,8-bis(thiophene-2-yl)-1,10-phenanthroline is 1:1.5~2.5.

[0013] Preferably, the coordination reaction is carried out at a temperature of 40-60°C for 6-12 hours.

[0014] Preferably, after the ion exchange, the method further includes: removing the solvent from the obtained ion exchange feed solution to obtain a crude product; purifying the crude product by silica gel column chromatography to collect the target component; concentrating the target component to obtain a solid; and washing and drying the solid sequentially to obtain the multifunctional iridium(III) complex.

[0015] Preferably, the eluent for the silica gel column chromatography purification includes dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol is 100:1 to 20:1.

[0016] The present invention also provides the application of the multifunctional iridium(III) complex described in the above technical solution or the multifunctional iridium(III) complex prepared by the preparation method described in the above technical solution in the preparation of photosensitizers for tumor photodynamic therapy.

[0017] The present invention also provides the application of the multifunctional iridium(III) complex described in the above technical solution or the multifunctional iridium(III) complex prepared by the preparation method described in the above technical solution in the preparation of a drug, wherein the drug is a drug for the prevention and / or treatment of tumors.

[0018] The present invention also provides the application of the multifunctional iridium(III) complexes described in the above technical solutions or the multifunctional iridium(III) complexes prepared by the above technical solutions in the preparation of diagnostic reagents or kits.

[0019] This invention provides a multifunctional iridium(III) complex.

[0020] Compared with the prior art, the present invention has the following significant advantages and beneficial effects: "One-stop" multifunctional integration: The multifunctional iridium(III) complex (Mito-Ir) provided by this invention innovatively integrates multiple functions such as mitochondrial targeting, phosphorescence imaging, type I / II ROS generation, NADH photocatalytic oxidation and pyroptosis induction into a single molecule, realizing integrated diagnosis and treatment and breaking through the limitation of traditional photosensitizers with single functions.

[0021] Excellent hypoxia tolerance: The multifunctional iridium(III) complex of the present invention can not only efficiently generate type II ROS (singlet oxygen) under aerobic conditions, but also cut off the cell energy supply in hypoxic environments through type I photodynamic pathway (generating superoxide anions, etc.) and consuming NADH, thereby achieving powerful killing of hypoxic tumor cells and fundamentally overcoming the key bottleneck in the treatment of solid tumors.

[0022] Cell death mechanism: The multifunctional iridium(III) complex provided in this invention can specifically induce pyroptosis through photoactivation. This inflammatory and lytic death mechanism can effectively bypass and overcome apoptosis resistance in tumor cells, providing a novel approach for treating drug-resistant tumors.

[0023] Powerful immune activation effect: Pyroptosis induced by multifunctional iridium(III) complexes, accompanied by the release of a large number of damage-associated molecular patterns (DAMPs) and inflammatory factors, can efficiently stimulate immunogenic cell death (ICD), thereby activating dendritic cells and initiating a T cell-mediated systemic anti-tumor immune response, which not only eliminates tumors in situ, but also has the potential to inhibit tumor metastasis and recurrence.

[0024] Synergistic effect and high safety: Mitochondrial targeting concentrates the destructive power of reactive oxygen species on the cell's "powerhouse," maximizing the killing efficiency; at the same time, its inherent phosphorescence properties can be used to monitor the location and distribution of drugs in cells in real time, providing the possibility of achieving precise and controllable photodynamic therapy, with both high efficacy and high potential safety. Attached Figure Description

[0025] Figure 1 This is a synthetic route diagram for preparing the multifunctional iridium(III) complex (Mito-Ir) in Example 1; Figure 2 The proton NMR spectrum of a multifunctional iridium(III) complex; Figure 3 The carbon NMR spectrum of a multifunctional iridium(III) complex; Figure 4 High-resolution mass spectrum of multifunctional iridium(III) complex; Figure 5 High-resolution mass spectra of multifunctional iridium(III) complexes (comparison of measured and theoretical simulations); Figure 6 This is the HPLC spectrum of a multifunctional iridium(III) complex; Figure 7 The ultraviolet-visible absorption spectrum and room-temperature phosphorescence emission spectrum of multifunctional iridium(III) complexes; Figure 8The image shows the detection results of type II ROS generated by the multifunctional iridium(III) complex (Mito-Ir) under illumination; Figure 9 The image shows the detection results of type I ROS generated by the multifunctional iridium(III) complex (Mito-Ir) under illumination; Figure 10 The image shows the detection results of NADH oxidation by the multifunctional iridium(III) complex (Mito-Ir) under light irradiation; Figure 11 The image shows the detection results of the reduction of cytochrome C by the multifunctional iridium(III) complex (Mito-Ir) under light irradiation; Figure 12 Confocal microscopy image of live-cell uptake of the multifunctional iridium(III) complex (Mito-Ir); Figure 13 Three-dimensional reconstructed image of live-cell uptake of the multifunctional iridium(III) complex (Mito-Ir); Figure 14 Subcellular localization confocal microscopy image of the multifunctional iridium(III) complex (Mito-Ir); Figure 15 Subcellular localization streak intensity analysis diagram for the multifunctional iridium(III) complex (Mito-Ir); Figure 16 Results of the cytotoxicity of the multifunctional iridium(III) complex (Mito-Ir) to EMT6 cells under light and dark conditions; Figure 17 Double staining of live / dead cells for the multifunctional iridium(III) complex (Mito-Ir); Figure 18 This is a verification diagram showing how multifunctional iridium(III) complex (Mito-Ir) combined with light treatment induces pyroptosis in tumor cells. Figure 19 This image shows the in vivo antitumor therapeutic effect of the multifunctional iridium(III) complex (Mito-Ir) in a tumor-bearing mouse model. Detailed Implementation

[0026] This invention provides a multifunctional iridium(III) complex, which is composed of an iridium(III) complex cation unit with the structure shown in Formula I and a corresponding coordinating anion: Formula I.

[0027] In this invention, the coordinating anion preferably includes [PF6]. - [BF4] - Or [ClO4] - [PF6] is further preferred.- .

[0028] The multifunctional iridium(III) complex provided by this invention is a cationic iridium(III) complex formed by co-coordinating a π-extended o-phenanthroline derivative, 3,8-bis(thiophene-2-yl)-1,10-phenanthroline, with a donor-acceptor-donor (DAD) structure as the main ligand and a cyclometalated phenylpyridine co-ligand, Ir2(ppy)4Cl2. The core innovation of this multifunctional iridium(III) complex lies in its compact size, resembling a "Swiss Army knife," yet integrating multiple functions: it not only efficiently targets mitochondria but also possesses the ability to perform phosphorescence imaging, generate type I and type II reactive oxygen species (ROS), and photocatalytically oxidize nicotinamide adenine dinucleotide (NADH). Under light irradiation, the multifunctional iridium(III) complex induces severe mitochondrial dysfunction through the aforementioned synergistic effects, thereby specifically activating the caspase-3 / GSDME signaling pathway and significantly inducing pyroptosis. This pyroptosis-based cell death mechanism can effectively overcome tumor cell apoptosis tolerance and is accompanied by the release of a large number of cytokines and damage-associated molecular patterns (DAMPs), triggering immunogenic cell death and activating the body's anti-tumor immune response. Therefore, this invention provides a lead compound for the development of novel pyroptosis inducers based on metal complexes, which has broad application prospects in the field of tumor therapy.

[0029] The present invention also provides a method for preparing the multifunctional iridium(III) complex described in the above technical solution, comprising the following steps: Ir2(ppy)4Cl2 having the structure shown in Formula a and 3,8-bis(thiophene-2-yl)-1,10-phenanthroline were dissolved in an organic solvent and subjected to a coordination reaction. An ion exchange reagent containing anion was added to the obtained coordination reaction solution to carry out ion exchange, thereby obtaining the multifunctional iridium(III) complex. Formula a.

[0030] Unless otherwise specified, the raw materials used in this invention are preferably commercially available products.

[0031] In this invention, the 3,8-bis(thiophene-2-yl)-1,10-phenanthroline has the structure shown in formula b: Formula b.

[0032] In this invention, the organic solvent preferably includes dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is preferably 1:1.

[0033] In this invention, the molar ratio of Ir2(ppy)4Cl2 to 3,8-bis(thiophene-2-yl)-1,10-phenanthroline is preferably 1:1.5 to 2.5, more preferably 1:2. In this invention, the preferred molar ratio of Ir2(ppy)4Cl2 to the organic solvent is 99 μmol:50 mL.

[0034] In this invention, the temperature of the coordination reaction is preferably 40-60°C, more preferably 50°C; the time is preferably 6-12 hours, more preferably 10 hours. In this invention, the coordination reaction is preferably carried out under a protective atmosphere, preferably nitrogen or argon. In this invention, the coordination reaction is preferably carried out under reflux and stirring conditions. In this invention, the stirring speed is preferably 400-600 rpm, more preferably 500 rpm.

[0035] After the coordination reaction, the present invention preferably further includes: cooling to room temperature to obtain the coordination reaction solution.

[0036] In this invention, the ion exchanger preferably includes ammonium hexafluorophosphate (NH4PF6), sodium tetrafluoroborate (NaBF4), or sodium perchlorate (NaClO4). In this invention, the molar ratio of the ion exchanger to Ir2(ppy)4Cl2 is preferably 2.0~2.5:1, specifically preferably 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, or 2.5:1. In this invention, the ion exchange temperature is preferably room temperature, i.e., neither additional heating nor cooling is required; the ion exchange time is preferably 0.5~1.5 h, more preferably 1 h; the ion exchange is preferably carried out under stirring conditions, and the stirring speed is preferably 400~600 rpm, more preferably 500 rpm.

[0037] Following the ion exchange, the present invention preferably further includes: removing the solvent from the obtained ion exchange feed solution to obtain a crude product; purifying the crude product by silica gel column chromatography to collect the target component; concentrating the target component to obtain a solid; and washing and drying the solid sequentially to obtain the multifunctional iridium(III) complex. In this invention, the eluent for the silica gel column chromatography purification preferably includes dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is preferably 100:1 to 20:1. In this invention, the target component is preferably a predominantly red band; the concentration is preferably rotary evaporation.

[0038] The present invention also provides the application of the multifunctional iridium(III) complex described in the above technical solution or the multifunctional iridium(III) complex prepared by the preparation method described in the above technical solution in the preparation of photosensitizers for tumor photodynamic therapy.

[0039] The present invention does not specifically limit the method of applying the multifunctional iridium(III) complex to prepare photosensitizers for tumor photodynamic therapy; any operation known to those skilled in the art can be used.

[0040] The present invention also provides the application of the multifunctional iridium(III) complex described in the above technical solution or the multifunctional iridium(III) complex prepared by the preparation method described in the above technical solution in the preparation of a drug, wherein the drug is a drug for the prevention and / or treatment of tumors.

[0041] This invention does not specify the method of applying the multifunctional iridium(III) complex to the preparation of drugs; any operation well known to those skilled in the art can be used. In a specific embodiment of this invention, the tumor is specifically a breast tumor. In this invention, when the drug is used to prevent or treat tumors, the concentration of the drug, calculated as a multifunctional iridium(III) complex, is preferably 1-10 mg / kg, more preferably 5 mg / kg.

[0042] The present invention also provides the application of the multifunctional iridium(III) complexes described in the above technical solutions or the multifunctional iridium(III) complexes prepared by the above technical solutions in the preparation of diagnostic reagents or kits.

[0043] The present invention does not impose specific limitations on the method of applying the multifunctional iridium(III) complex to the preparation of diagnostic reagents or kits; any operation known to those skilled in the art can be used.

[0044] The following detailed description, in conjunction with embodiments, illustrates the multifunctional iridium(III) complexes, their preparation methods, and applications provided by this invention. However, these descriptions should not be construed as limiting the scope of protection of this invention.

[0045] Example 1 Synthesis of the multifunctional iridium(III) complex Mito-Ir according to Figure 1 The synthetic route shown is for the preparation of multifunctional iridium(III) complexes (Mito-Ir), specifically as follows: Ir2(ppy)4Cl2 (106 mg, 99 μmol) and 3,8-bis(thiophene-2-yl)-1,10-phenanthroline (68 mg, 198 μmol) were dissolved in 50 mL of a 1:1 mixture of dichloromethane and methanol. The reaction mixture was subjected to a coordination reaction at 50 °C under reflux, stirring (500 rpm), and nitrogen atmosphere for 10 h. After the coordination reaction was completed, the resulting reaction mixture was cooled to room temperature, and then ammonium hexafluorophosphonate (32 mg, 198 μmol) was added. The solution was stirred at 500 rpm for 1 h for ion exchange. After the ion exchange was completed, the solvent in the ion exchange solution was removed to obtain the crude product. The crude product was purified by silica gel column chromatography with a gradient elution of dichloromethane / methanol (v / v, 100:1~20:1). The main red band was collected, the solvent was removed by rotary evaporation, and the product was washed and dried to obtain 180.2 mg of pure red solid multifunctional iridium(III) complex (denoted as Mito-Ir complex), with a yield of 91.9%.

[0046] Test Example 1 By nuclear magnetic resonance hydrogen spectrum ( 1 H NMR, carbon spectrum ( 13 The structure and purity of the product were confirmed by 12C NMR, high-resolution mass spectrometry (HR-MS), and high-performance liquid chromatography (HPLC).

[0047] Figure 2 This is the 1H NMR spectrum of a multifunctional iridium(III) complex. Figure 3 This is the carbon NMR spectrum of a multifunctional iridium(III) complex. Figure 4 This is a high-resolution mass spectrum of a multifunctional iridium(III) complex. Figure 5 High-resolution mass spectra of multifunctional iridium(III) complexes (comparison of measured and theoretical simulations). Figure 6 The HPLC spectrum of a multifunctional iridium(III) complex is shown below. Figures 2-5 It can be seen that the experimental molecular weight of Mito-Ir is completely consistent with the theoretical calculation, confirming the successful synthesis of the target complex; from Figure 6 It can be seen that the retention time of Mito-Ir is 17.696 minutes, and the purity is as high as 99.13%.

[0048] Test Example 2 Characterization of photophysical and photochemical properties of multifunctional iridium(III) complexes (1) Photophysical properties of the multifunctional iridium(III) complex Mito-Ir UV-Vis absorption and phosphorescence spectroscopy: Mito-Ir was dissolved in water, and its UV-Vis absorption spectrum and room-temperature phosphorescence emission spectrum were measured. The results are as follows: Figure 7As shown, A is the ultraviolet-visible absorption spectrum, and B is the room-temperature phosphorescence emission spectrum. Figure 7 It can be seen that Mito-Ir has a significant metal-ligand charge transfer absorption band in the visible light region (>400nm), which is beneficial for excitation by blue light with good penetration into biological tissues; at the same time, it shows strong red phosphorescence emission at ~630nm, which is suitable for biological imaging.

[0049] (2) Determination of reactive oxygen species (ROS) generation capacity 2.1 Type II ROS (singlet oxygen, 1 O2: 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA) is used as the singlet oxygen ( 1 The photosensitizer is a specific trapping agent for O2, and its type II photodynamic activity is quantitatively assessed by monitoring the decay of its characteristic absorption peak at 378 nm. The principle is based on... 1 O2 can undergo a [4+2] cycloaddition reaction with the anthracene ring of ABDA, causing the intensity of this characteristic absorption peak to decrease with increasing light exposure time, and the degradation rate is related to the amount of oxygen in the system. 1 The O2 production is directly proportional. The specific experimental procedure is as follows: Aqueous solutions containing Mito-Ir (10 μM) or the reference [Ru(bpy)3]Cl2 (10 μM) and ABDA (50 μM) are placed separately in quartz cuvettes, using a 410 nm blue LED light source (power density 20 mW / cm²). 2 The solution was irradiated, and the absorbance was measured using a UV-Vis spectrophotometer every 10 seconds. The change in absorbance at 378 nm was recorded. The results are as follows: Figure 8 As shown, A is the UV-Vis absorption spectrum of ABDA as a function of illumination time (hv) in the presence of Mito-Ir; B is the UV-Vis absorption spectrum of ABDA as a function of illumination time (hv) in the presence of [Ru(bpy)3]Cl2; and C is a comparison of the kinetic curves of Mito-Ir+ABDA+hv and [Ru(bpy)3]Cl2+ABDA+hv at 378 nm based on the time-varying Ln(A / A0). Figure 8 As can be seen from A and B in the diagram, under continuous illumination, the intensity of the characteristic absorption peak of ABDA in both systems decreases in a time-dependent manner, indicating that both systems exhibit [a certain characteristic]. 1 O2 generation; by plotting the normalized absorbance ratio (A / A0) over time and performing kinetic analysis (e.g.) Figure 8 As shown in C), the ABDA degradation rate constant of the Mito-Ir group was found to be significantly higher than that of the [Ru(bpy)3]Cl2 reference group, confirming that Mito-Ir has a superior singlet oxygen generation capacity.

[0050] 2.2 Type I ROS (Superoxide Anion and Hydroxyl Radical): For the assessment of Type I photodynamic activity, dihydrorhodamine 123 (DHR123, 5 μM) and hydroxyphenyl fluorescein (HPF, 5 μM) were used in parallel as superoxide anions (O2• - Specific fluorescent probes for hydroxyl radicals (·OH) and hydroxyl radicals were developed. The experimental system consisted of an aqueous solution containing Mito-Ir or [Ru(bpy)3]Cl2 (both at a concentration of 10 μM) and the corresponding probe. Under the same 410 nm light source irradiation conditions, emission spectra at 500-600 nm were collected immediately after 10 seconds or 1 minute of irradiation using a fluorescence spectrometer. The characteristic emission peaks of DHR123 and HPF were located near 525 nm and 515 nm, respectively. The results are as follows: Figure 9 As shown, A is the fluorescence emission spectrum of DHR123 with illumination (hv) time in the presence of Mito-Ir; B is the fluorescence emission spectrum of DHR123 with illumination (hv) time in the presence of [Ru(bpy)3]Cl2; C is the kinetic curve of the normalized fluorescence intensity (I / I0) at 525 nm as a function of time for the corresponding systems in A and B; D is the fluorescence emission spectrum of HPF with illumination (hv) time in the presence of Mito-Ir; E is the fluorescence emission spectrum of HPF with illumination (hv) time in the presence of [Ru(bpy)3]Cl2; and F is the kinetic curve of the normalized fluorescence intensity (I / I0) at 515 nm as a function of time for the corresponding systems in D and E. Figure 9 The results show that with prolonged illumination time, the fluorescence intensity of Mito-Ir and the two probe coexisting systems at their respective characteristic emission wavelengths is rapidly and significantly enhanced. The enhancement magnitude and rate are significantly greater than those of the [Ru(bpy)3]Cl2 control group tested under the same conditions, thus confirming that Mito-Ir can efficiently catalyze the generation of superoxide anions and hydroxyl radicals under illumination, exhibiting significant type I photodynamic activity.

[0051] Test Example 3 Photocatalytic oxidation of NADH β-Nicotinamide adenine dinucleotide (NADH) was used as a model substrate for coenzyme I. The photocatalytic oxidation ability of the photosensitizer was evaluated by monitoring the decay of its characteristic absorption peak at 339 nm. NADH exhibits characteristic absorption at this wavelength, while its oxidation product NAD+ shows no significant absorption. Therefore, the rate of decrease in absorbance directly reflects the oxidation efficiency of NADH. The specific steps were as follows: Mito-Ir (10 μM) or the reference [Ru(bpy)3]Cl2 (10 μM) and NADH (100 μM) were co-dissolved in an aqueous solution, with a total volume of 2 mL. The solution was placed in a quartz cuvette, and a 410 nm blue LED light source (power density 20 mW / cm²) was used. 2The solution was vertically irradiated, and the irradiation was paused after every 5 seconds. Immediately afterward, the absorption spectrum from 230 to 570 nm was scanned using a UV-Vis spectrophotometer, and the change in absorbance at 339 nm was recorded. The results are shown in […]. Figure 10 In the figure, A represents the UV-Vis absorption spectrum of NADH as a function of illumination time (hv) in the presence of Mito-Ir; B represents the UV-Vis absorption spectrum of NADH as a function of illumination time (hv) in the presence of [Ru(bpy)3]Cl2; and C represents a comparison of the kinetic curves of Mito-Ir+NADH+hv and [Ru(bpy)3]Cl2+NADH+hv at 339 nm based on the time-varying ln(A / A0). The results are as follows: Figure 10 As shown, under illumination, the absorption peak intensity of NADH at 339 nm in the Mito-Ir-containing system continuously decreased with illumination time, indicating that NADH was effectively oxidized; while the control group containing [Ru(bpy)3]Cl2 showed no significant change, proving that Mito-Ir has the ability to efficiently catalyze the oxidation of NADH under illumination, revealing its potential to enhance the effect of photodynamic therapy by intervening in cellular energy metabolism.

[0052] Furthermore, to investigate photocatalytic redox cascades related to cellular energy metabolism, the effects of Mito-Ir in the presence of oxidized cytochrome c (Fe2+) were studied. 3+ The role of α-cyt c (10 μM) in the system of NADH (10 μM). Under photocatalytic conditions, the excited-state photocatalyst can promote the transfer of electrons from NADH to Fe. 3+ -cyt c transfer, thereby oxidizing NADH to NAD. + At the same time, Fe 3+ -cyt c is reduced to Fe 2+ -cyt c, the latter has a characteristic absorption peak at 550 nm. If this process occurs intracellularly, it can consume NADH and perturb the electron transport chain, leading to cellular energy metabolism disorders. The experimental method is as follows: Mito-Ir (1 μM) and Fe 3+ -cyt c (10 μM) and NADH (10 μM) were co-dissolved in an aqueous solution and analyzed under the same conditions (410 nm, 20 mW / cm²). 2 Irradiation was performed, and the absorption spectrum in the range of 230-700 nm was measured after every minute of irradiation, with a focus on monitoring the change in absorbance at 550 nm. The results are shown in […]. Figure 11In the figure, A represents the UV-Vis absorption spectrum of the NADH-cytochrome c (cyt c) coexisting system in the presence of Mito-Ir as a function of light exposure time (hv); B represents the UV-Vis absorption spectrum of the NADH-cytochrome c (cyt c) coexisting system in the presence of [Ru(bpy)3]Cl2 as a function of light exposure time (hv); and C represents a comparison of the kinetic curves of Mito-Ir+NADH+cyt c+hv and [Ru(bpy)3]Cl2+NADH+cyt c+hv at 550 nm based on the change of ln(A / A0) over time. The results are as follows: Figure 11 As shown, under illumination, the absorption intensity of the Mito-Ir-containing system at 550 nm decreases significantly over time, indicating that Fe... 3+ -cyt c was effectively reduced; while the [Ru(bpy)3]Cl2 control group showed no significant change. These results demonstrate that Mito-Ir can photocatalyze the oxidation of NADH and drive the reduction of cytochrome c, constructing a cascade reaction that consumes intracellular reducing equivalents and disrupts the respiratory chain, showing potential application value in overcoming the challenges of tumor hypoxia-related treatments.

[0053] Test Example 4 Cellular uptake and mitochondrial colocalization The ability of photosensitizers to achieve efficient intracellular delivery and precise subcellular localization is crucial for their therapeutic efficacy. To investigate the cellular uptake behavior and subcellular distribution characteristics of Mito-Ir, this study conducted systematic live-cell confocal microscopy imaging analysis. Specifically, cells were co-incubated with Mito-Ir (10 μM) at 37°C, and images were taken using confocal microscopy at different time points (0.25 h, 0.5 h, 1 h, 2 h). The results are shown in [Figure number missing]. Figure 12 .from Figure 12 It can be seen that Mito-Ir can rapidly enter EMT6 cells, and significant intracellular accumulation can be observed within 2 hours of incubation.

[0054] To further investigate the three-dimensional spatial distribution of Mito-Ir within cells, three-dimensional reconstructed images were generated. The specific procedure involved Z-stack scanning of the cells and subsequent three-dimensional reconstruction using software. The results are shown below. Figure 13 ,like Figure 13 As shown, the three-dimensional reconstructed images confirm that Mito-Ir was successfully internalized into the cell, and its XY cross-sectional images show a typical punctate fluorescence distribution, suggesting that it may be located in a specific subcellular organelle.

[0055] To further clarify the subcellular localization of Mito-Ir, co-localization experiments were performed. Cells were co-incubated with Mito-Ir (10 μM) for 30 minutes, then stained with mitochondrial red fluorescent probe (MitoTracker Red, MTR) and lysosomal green fluorescent probe (LysoTracker Green, LTG), respectively, and confocal imaging was performed. The results are shown in the figure. Figure 14 , Figure 14 The results showed that the phosphorescence signal of Mito-Ir highly overlapped with the fluorescence signal of the mitochondrial probe, with a Pearson correlation coefficient (PCC) of 0.91; while the colocalization with the lysosomal probe was relatively low (PCC = 0.32). Fluorescence intensity curve analysis of selected regions further confirmed this result.

[0056] To further clarify, regarding Figure 14 The fluorescence intensity of Mito-Ir and two subcellular fluorescent probes was analyzed by streaking, i.e., a straight line was drawn along a specific region of the cell, and the fluorescence intensity distribution of each channel along the line was extracted. The results are shown in [Figure number missing]. Figure 15 ,like Figure 15 As shown, the signal peak of Mito-Ir is highly consistent with the mitochondrial probe signal, but has almost no overlap with the lysosomal probe signal.

[0057] The remarkable mitochondrial targeting exhibited by Mito-Ir can be attributed to its integrative molecular design: moderate lipophilicity (log P). o The α-hydroxyl content ( / w = 1.39 ± 0.02) facilitates its crossing of biological membranes, while its delocalized cation properties, driven by the mitochondrial membrane potential (ΔΨm), lead to its specific enrichment in the mitochondrial matrix via electrophoresis. This efficient and rapid cellular uptake and precise mitochondrial localization capability lays a solid foundation for Mito-Ir to induce immediate mitochondrial dysfunction under light irradiation and achieve highly efficient mitochondrial-targeted photodynamic therapy.

[0058] Test Example 5 The MTS assay was used to evaluate the cytotoxicity of Mito-Ir in EMT6 cells. For the dark toxicity assay of Mito-Ir, EMT6 cells were used at a concentration of 1×10⁻⁶. 4EMT6 cells were seeded at a density of 1 cell / well in 96-well plates and cultured for 24 hours. Then, different concentrations (0, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 20, 30, 40, 50, 75, 125, 200 µM) of Mito-Ir were added to each well. After 48 hours of incubation, 10 µL of MTS solution (CellTiter 96 AQueous One Solution Cell Proliferation Assay) was added to each well, and incubation continued for 3 hours under the same conditions. Finally, the absorbance at 500 nm was measured using a SpectraMax 190 microplate reader. For phototoxicity experiments, EMT6 cells were incubated with different concentrations of Mito-Ir for 0.5 hours, followed by incubation under a 410 nm LED lamp (20 mW·cm⁻¹). -2 Irradiate for 30 minutes, then continue incubation for 41 hours. Afterwards, add 10 µL of MTS solution and continue incubation for 3 hours. The absorbance at 500 nm is measured using a SpectraMax 190 microplate reader; the results are shown below. Figure 16 ,like Figure 16 As shown, Mito-Ir exhibits certain dark toxicity to EMT6 cells, IC50... 50 The calculated value is 1.48 µM. However, this is in contrast to the extremely phototoxic IC. 50 The value is 4.8 nM, and the phototoxicity index is as high as 308.

[0059] To verify the photoactivated antitumor effect at the cellular level, a live / dead cell double staining assay (Ca-AM / PI) was further used to visually assess cell status. Calcein (Ca-AM) dye stained live cells, exhibiting green fluorescence; while propidium iodide (PI) dye stained dead cells, exhibiting red fluorescence. This allowed for a direct distinction and analysis of cell viability. The results are shown in [Figure number missing]. Figure 17 .like Figure 17 As shown, cells treated with different methods were stained with both Ca-AM and PI. Only the Mito-Ir+hv group showed a strong PI signal (red) and a weak Ca-AM signal (green), while the other groups (control group, light-only group, and Mito-Ir-only group) showed a strong Ca-AM signal (green) and a weak PI signal (red). The experimental results confirm that Mito-Ir can kill tumor cells under light irradiation.

[0060] Test Example 6 Cell death mechanism research EMT6 cells were co-incubated with Mito-Ir (10 µM) for 2 hours. Subsequently, the cells were washed three times with PBS, and then exposed to light or dark conditions, respectively. Cell lysis buffer was added, and the cells were collected. Next, total protein was extracted and analyzed by Western blotting (WB). After electrophoresis, the samples were transferred to a polyvinylidene fluoride (PVDF) membrane, blocked with 5% skim milk, and then incubated overnight at 4°C with primary antibody. Following this, the membrane was incubated for 1 hour at room temperature with horseradish peroxidase (HRP)-labeled secondary antibody. Finally, the images were developed using an enhanced chemiluminescence reagent. The results are shown in the figure below. Figure 18 .like Figure 18 As shown, in cells treated with Mito-Ir+ light (hv), the expression of activated caspase-3 and its cleavage product GSDME-N fragment was significantly upregulated, confirming that pyroptosis is carried out through the caspase-3 / GSDME pathway.

[0061] Test Example 7 Evaluation of antitumor efficacy in animals at the in vivo level This embodiment uses a mouse tumor model to evaluate the in vivo therapeutic effect of Mito-Ir.

[0062] Model establishment: 32 female Balb / c mice (weighing 18-20g, 8 weeks old) were subcutaneously injected with 25µL of EMT6 cell suspension (1×10⁻⁶ cells) on the right posterior back. 6 (1 cell / each). After 7 days, tumors with a volume of approximately 100 mm were selected. 3 The mice were randomly divided into 4 groups of 8 mice each for subsequent experiments.

[0063] EMT6 tumor-bearing mice were randomly divided into 4 groups (n=8 per group) and received different treatments: (1) Control group, (2) Light irradiation only group (hv), (3) Mito-Ir treatment only group (Mito-Ir), and (4) Mito-Ir + light irradiation group (Mito-Ir + hv). Mito-Ir treatment: Mito-Ir (5 mg·kg⁻¹) was injected into the tumor. -1 Irradiation conditions: 410nm, power density 20mW·cm⁻¹ -2 The treatment lasted for 60 minutes. During the treatment period, the mice's body weight and tumor volume were measured every two days for a total of 14 days. The tumor volume was calculated using the formula: Volume = 0.5 × Length × Width 2 After 14 days of treatment, the mice were euthanized, the tumors were removed, weighed, and photographed. The results are shown below. Figure 19 ,like Figure 19 As shown, compared with the control group and the dark group, the tumor growth of mice in the Mito-Ir+hv group was significantly inhibited, and some mice had complete tumor regression after treatment.

[0064] Conclusion: The above embodiments fully demonstrate that the multifunctional iridium(III) complex (Mito-Ir) provided by the present invention has excellent targeting, imaging, multimodal photodynamic killing and immune activation functions, and has extremely high application value in the field of tumor photodynamic therapy.

[0065] This invention provides a novel and high-performance photosensitizer: (1) A cationic iridium(III) complex (Mito-Ir) with a π-extended o-phenanthroline derivative having a "donor-receptor-donor" (DAD) structure as the main ligand is designed and synthesized. (2) Achieving efficient organelle targeting and integrated diagnosis and treatment: The multifunctional iridium(III) complex can specifically target the mitochondria of tumor cells and utilize its inherent strong phosphorescence emission properties to achieve real-time, in-situ imaging of target cells and subcellular structures, providing visual guidance for treatment. (3) Overcoming the limitations of the tumor hypoxic microenvironment: The multifunctional iridium(III) complex is endowed with the ability to synergistically generate type I and type II reactive oxygen species (ROS). Especially under hypoxic conditions, through efficient type I photodynamic processes (such as the generation of superoxide anion free radicals) and the ability to photocatalytically oxidize NADH, the cell energy metabolism is cut off, ensuring that a strong oxidative stress killing effect can still be generated in the harsh tumor microenvironment. (4) Opening up new cell death pathways to overcome treatment resistance: Utilizing the severe mitochondrial dysfunction induced by this multifunctional iridium(III) complex, the caspase-3 / GSDME signaling pathway is specifically activated, and pyroptosis is induced dominantly, thereby effectively avoiding and overcoming the problem of tumor treatment resistance caused by apoptosis resistance. (5) Stimulating the body's anti-tumor immune response: Through the pyroptosis successfully induced by the multifunctional iridium(III) complex, strong immunogenic cell death (ICD) is triggered, releasing a large number of damage-associated molecular patterns (DAMPs) and inflammatory factors, activating dendritic cells and T cells, and finally establishing a durable and systematic anti-tumor immune surveillance and attack capability. (6) Finally, providing a comprehensive treatment platform with "one dose, multiple effects": Integrating the above-mentioned functions such as targeting, imaging, multimodal ROS generation, energy metabolism intervention, pyroptosis induction and immune activation into a single multifunctional iridium(III) complex (Mito-Ir), a high-efficiency, synergistic, and high-performance photodynamic therapy platform that can overcome multidrug resistance is constructed, providing an innovative solution and lead compound for clinical tumor treatment.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A multifunctional iridium(III) complex, characterized in that, It is composed of the iridium(III) complex cationic unit with the structure shown in Formula I and the corresponding coordinating anion: Equation I.

2. The multifunctional iridium(III) complex according to claim 1, characterized in that, The coordinating anion includes [PF6]. - [BF4] - Or [ClO4] - .

3. The method for preparing the multifunctional iridium(III) complex according to claim 1 or 2, characterized in that, Includes the following steps: Ir2(ppy)4Cl2 having the structure shown in Formula a and 3,8-bis(thiophene-2-yl)-1,10-phenanthroline were dissolved in an organic solvent and subjected to a coordination reaction. An ion exchange reagent containing anion was added to the obtained coordination reaction solution to carry out ion exchange, thereby obtaining the multifunctional iridium(III) complex. Formula a.

4. The preparation method according to claim 3, characterized in that, The molar ratio of Ir2(ppy)4Cl2 to 3,8-bis(thiophene-2-yl)-1,10-phenanthroline is 1:1.5~2.

5.

5. The preparation method according to claim 3 or 4, characterized in that, The coordination reaction is carried out at a temperature of 40-60℃ for 6-12 hours.

6. The preparation method according to claim 3, characterized in that, After the ion exchange, the process further includes: removing the solvent from the obtained ion exchange feed solution to obtain a crude product; purifying the crude product by silica gel column chromatography to collect the target component; concentrating the target component to obtain a solid; and washing and drying the solid sequentially to obtain the multifunctional iridium(III) complex.

7. The preparation method according to claim 6, characterized in that, The eluent for the silica gel column chromatography purification includes dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol is 100:1 to 20:

1.

8. The use of the multifunctional iridium(III) complex according to claim 1 or 2 or the multifunctional iridium(III) complex prepared by the preparation method according to any one of claims 3 to 7 in the preparation of photosensitizers for tumor photodynamic therapy.

9. The use of the multifunctional iridium(III) complex according to claim 1 or 2 or the multifunctional iridium(III) complex prepared by any one of claims 3 to 7 in the preparation of a drug, wherein the drug is a drug for the prevention and / or treatment of tumors.

10. The use of the multifunctional iridium(III) complex according to claim 1 or 2 or the multifunctional iridium(III) complex prepared by the preparation method according to any one of claims 3 to 7 in the preparation of diagnostic reagents or kits.

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