A cyclometalated Ir(III) complex and its preparation method and application

Abstract

The present invention discloses a cyclometallated Ir(III) complex and a preparation method and application thereof. The structural formula of the cyclometallated Ir(III) complex of the present invention is as follows: The cyclometallated Ir(III) complex of the present invention has good photophysical properties and photocatalytic activity, can exert type I and type II photodynamic effects, efficiently generate reactive oxygen species, and has the characteristics of endoplasmic reticulum targeting and synergistic enhancement of anti-tumor immune regulation, and has excellent anti-tumor effect when applied to anti-tumor photodynamic therapy agents.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor drugs, and more specifically to a cyclometalated Ir(III) complex and a preparation method and application thereof. Background Art

[0002] Oral squamous cell carcinoma (OSCC) is formed by the malignant transformation of oral squamous epithelium. The traditional treatment methods are mainly surgical treatment, adjuvant radiotherapy or chemotherapy. Among them, platinum drugs represented by cisplatin are the most widely used metal complex anti-tumor drugs in clinical practice, but they have serious toxic side effects and drug resistance. Iridium, which belongs to the platinum group elements, not only has similar properties to platinum, but also has excellent photophysical properties, intracellular reaction catalysis ability and unique subcellular organelle localization. It also has an anti-tumor mechanism different from traditional platinum drugs, making cyclometalated iridium metal complexes a very potential new type of high-efficiency, low-toxicity, targeted anti-tumor drug after platinum drugs.

[0003] Photodynamic therapy (PDT) is a technology that uses the photodynamic effect to treat diseases. It is an effective clinical treatment method for superficial tumors (such as esophageal cancer, bladder cancer, and malignant melanoma). Its mechanism of action is that the photosensitizer taken up by the tissue is excited by laser irradiation of a specific wavelength, and catalyzes the oxygen inside the tumor through energy transfer to generate cytotoxic reactive oxygen species (ROS, such as singlet oxygen, hydroxyl free radicals, superoxide anions, etc.), which then destroys the structure and function of cells and organelles, causing cell death, and achieving the purpose of treating tumors. Compared with traditional therapies, photodynamic therapy is a non-invasive, low-toxic, and targeted treatment option.

[0004] Cyclometallated iridium complexes have been reported to be able to induce cell ferroptosis under hypoxic conditions through the type I photodynamic pathway as small molecule photosensitizers, activating tumor immune responses. For example, the prior art discloses a metal iridium (III) complex and its preparation method and application. The anti-tumor metal iridium (III) complex can be used as a photosensitizer to participate in photodynamic therapy, especially in human non-small cell lung cancer cells, with a very high phototoxicity index, and is an efficient photodynamic diagnostic and therapeutic agent targeted at lung cancer; however, the anti-tumor metal iridium (III) complex has limited subcellular organelle targeting, especially insufficient endoplasmic reticulum targeting, and low tumor immunogenicity activation efficiency, which hinders its clinical application in oral squamous cell carcinoma. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects and deficiencies of the endoplasmic reticulum targeting and tumor immunogenicity activation efficiency of the existing anti-tumor metal iridium (III) complexes, and to provide a cyclometalated Ir (III) complex.

[0006] Another object of the present invention is to provide a method for preparing the above cyclometalated Ir(III) complex.

[0007] Another object of the present invention is to provide the use of the above-mentioned cyclometalated Ir(III) complex in the preparation of anti-tumor materials or anti-tumor drugs.

[0008] Another object of the present invention is to provide an anti-tumor photodynamic therapy agent comprising the above-mentioned cyclometalated Ir(III) complex.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention protects a cyclometalated Ir(III) complex, the structural formula of which is shown in formula (I):

[0011]

[0012] The cyclometallated Ir(III) complex of the above structure of the present invention has good photophysical properties and photocatalytic activity, can exert type I and type II photodynamic effects, and efficiently generate reactive oxygen; at the same time, by introducing aromatic long-chain compound segments containing β-phenylethylamine, the endoplasmic reticulum targeting is enhanced, thereby synergistically enhancing the anti-tumor immune regulation characteristics, and is a new type of metal anti-tumor photodynamic therapy agent with good biosafety.

[0013] The present invention also protects a method for preparing the above-mentioned cyclometalated Ir(III) complex, comprising the following steps:

[0014] The bridged iridium precursor [Ir(ppy)2Cl]2 and the ligand L are mixed and dispersed in an organic solvent, reacted at 50-70°C for 16-32h under the protection of an inert gas, and purified to obtain a cyclometalated Ir(III) complex;

[0015] Wherein, the structural formula of the ligand L is shown in formula (II):

[0016]

[0017] Preferably, the reaction temperature is 60° C. and the reaction time is 24 h.

[0018] Specifically, the molar ratio of the bridged iridium precursor [Ir(ppy)2Cl]2 to the ligand L is 1:(2.0-2.5). Optionally, the molar ratio of the bridged iridium precursor [Ir(ppy)2Cl]2 to the ligand L is 1:2.0, 1:2.1, or 1:2.3.

[0019] In a specific embodiment, the organic solvent is a mixture of CH2Cl2 and CH3OH in a volume ratio of (1-3): 1. Preferably, the volume ratio of CH2Cl2 to CH3OH is 2:1.

[0020] Specifically, the detailed steps of the above preparation method are as follows:

[0021] A suspension of the bridged iridium precursor [Ir(ppy)2Cl]2 (200.8 mg, 179.47 μmol) and a ligand L (80.0 mg, 200.8 μmol) were mixed and dispersed in an organic solvent (CH2Cl2 / CH3OH, v / v=2 / 1), and the mixture was refluxed at 60°C for 24 h under nitrogen protection. The reaction mixture was cooled, and the solvent was removed by rotary evaporation. N,N-dimethylformamide (DMF, 2 mL) and a saturated potassium hexafluorophosphate solution (KPF6, 50 ml) were added to dissolve the solid sample, and the sample was filtered and rinsed with water to obtain a solid sample. The solid was purified by rapid column chromatography on a silica gel column (CH3CN / H2O, v / v=10 / 1), and the solvent was removed by evaporation under nitrogen. The product was a yellow solid, which was the cyclometalated Ir(III) complex.

[0022] Specifically, the ligand L can be prepared by the following preparation method:

[0023] The reaction monomer I, O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (CAS No. 148893-10-1), N,N′-diisopropylethylamine (CAS No. 7087-68-5) and 1,10-phenanthroline-5-amino (CAS No. 54258-41-2) were mixed evenly, reacted at 40-50° C. for 6-10 h, and purified to obtain the ligand L;

[0024] Wherein, the structural formula of the reaction monomer I is shown in formula (III):

[0025]

[0026] Preferably, the reaction temperature is 45° C. and the reaction time is 8 h.

[0027] Specifically, the molar ratio of the reaction monomer I to 1,10-phenanthroline-5-amino group is 1:(1.0-1.2).

[0028] Specifically, the detailed steps of the above preparation method are as follows:

[0029] The reaction monomer I was added to O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU, 5.26 g, 13.56 mmol) and N,N′-diisopropylethylamine (DIPEA, 4.7 mL), stirred for about 30 min, then 1,10-phenanthroline-5-amino (882.4 mg, 4.52 mmol) was added, and refluxed at 45° C. for 8 h. The solvent was evaporated to remove, and then purified by rapid column chromatography on a silica gel column (CH3CN / H2O, v / v=10 / 1); in order to further improve the purity, recrystallization was performed with acetonitrile to obtain a white foam solid, which was the ligand L.

[0030] Optionally, the reactive monomer I can be prepared by the following preparation method:

[0031] β-phenylethylamine (CAS No. 64-04-0) and succinic anhydride (CAS No. 108-30-5) are dissolved in an organic solvent and reacted at 40-50° C. for 2-8 hours to obtain reaction monomer I. Preferably, the reaction temperature is 45° C. and the reaction time is 2 hours.

[0032] Specifically, the preparation method is as follows: β-phenylethylamine (0.6 mL) and succinic anhydride (500.0 mg, 5.00 mmol) are dissolved in CH2Cl2 (100 mL), and heated at 45°C for 2 h to obtain reaction monomer I.

[0033] The use of the above-mentioned cyclometalated Ir(III) complex in the preparation of anti-tumor materials or anti-tumor drugs is also within the protection scope of the present invention.

[0034] Specifically, the anti-tumor material is an anti-oral squamous cell carcinoma material, and the anti-tumor drug is an anti-oral squamous cell carcinoma drug.

[0035] The present invention also protects an anti-tumor photodynamic therapy agent comprising the cyclometallated Ir(III) complex.

[0036] Compared with the prior art, the present invention has the following beneficial technical effects:

[0037] The cyclometallated Ir(III) complex of the present invention has dark toxicity to cells comparable to that of cisplatin, and its phototoxicity index exceeds 100, has good biosafety, can be selectively taken up by tumor cells, further targets the endoplasmic reticulum of tumor cells, and produces an endoplasmic reticulum stress effect; and can generate a large amount of ROS under laser irradiation, exert a strong PDT effect, and effectively kill SCC1 oral cancer cells.

[0038] The cyclometallated Ir(III) complex of the present invention can also regulate the tumor immune microenvironment under light conditions. The induced endoplasmic reticulum stress can cause tumor cell immunogenic death (ICD), activate immune response through damage-associated molecular patterns (DAMPs), such as the expression of calreticulin, the secretion of high mobility group protein 1 (HMGB1), and the release of ATP molecules, and can cooperate with PD-L1 immune checkpoint blockade to inhibit tumor growth. It is an anti-tumor drug with outstanding photodynamic immunotherapy potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of the synthesis route of the cyclometalated Ir(III) complex in the present invention.

[0040] Figure 2 This is the electron spin resonance spectrum of the cyclometallated Ir(III) complex in Example 4.

[0041] Figure 3 This is the fluorescence emission spectrum of the titration of the cyclometalated Ir(III) complex and lecithin in Example 4.

[0042] Figure 4 The organelle localization of Ir was analyzed by confocal microscopy.

[0043] Figure 5 Flow cytometry was used to analyze the ROS production after Ir-PDT treatment.

[0044] Figure 6 Western blot analysis of the expression of eIF2α, p-eIF2α, and CHOP after Ir-PDT treatment.

[0045] Figure 7 Transmission electron microscopy analysis of cell morphology after Ir-PDT treatment.

[0046] Figure 8 Flow cytometry was used to analyze the expression of CRT after Ir-PDT treatment.

[0047] Fig. 9 Flow cytometry was used to analyze the expression of HMGB1 after Ir-PDT treatment.

[0048] Fig.10 The chemiluminescence method was used to analyze the ATP release after Ir-PDT treatment.

[0049] Fig.11 Western blot analysis of PD-L1 expression after Ir-PDT treatment.

[0050] Fig.12A CDX model was used to study the effect of Ir-mediated PDT on the treatment of oral cancer and its combination with PD-L1 inhibitors to synergistically enhance the anti-tumor immune response. DETAILED DESCRIPTION

[0051] The present invention is further described below in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the raw materials and reagents used in the embodiments of the present invention are conventionally purchased raw materials and reagents.

[0052] Example 1

[0053] The structural formula of the reactive monomer I is: It can be prepared by the following preparation method:

[0054] β-Phenylethylamine (0.6 mL) and succinic anhydride (500.0 mg, 5.00 mmol) were dissolved in CH2Cl2 (100 mL) and heated at 45°C for 2 h to obtain reaction monomer I.

[0055] Example 2

[0056] The structural formula of ligand L is: It can be prepared by the following preparation method:

[0057] β-Phenylethylamine (0.6 mL) and succinic anhydride (500.0 mg, 5.00 mmol) were dissolved in CH2Cl2 (100 mL) and heated at 45°C for 2 h to obtain reaction monomer I; then O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU, 5.26 g, 13.56 mmol) and N,N'-diisopropylethylamine (DIPEA, 4.7 mL) were added, stirred for about 30 min, and then 1,10-phenanthroline-5-amino (882.4 mg, 4.52 mmol) was added, and reflux was continued for 8 h. The solvent was evaporated and then purified by rapid column chromatography on a silica gel column (CH3CN / H2O, v / v=10 / 1). In order to further improve the purity, a white foam solid was obtained by recrystallization with acetonitrile, which was the ligand L.

[0058] Example 3

[0059] The structural formula of the bridged iridium precursor [Ir(ppy)2Cl]2 is: It can be prepared by the following preparation method:

[0060] Hydrated iridium trichloride and 2-phenylpyridine were mixed and dispersed in an organic solvent (2-Ethoxyethanol / H2O, v / v=3 / 1), and the reaction was refluxed at 80°C for 14 hours under nitrogen protection. After purification, the cyclometallated bridged iridium precursor [Ir(ppy)2Cl]2 was obtained.

[0061] Example 4

[0062] A cyclometalated Ir(III) complex, the structural formula of which is as follows:

[0063]

[0064] The above-mentioned cyclometallated Ir(III) complex can be prepared by the following preparation method (eg Figure 1 shown):

[0065] The suspension of the bridged iridium precursor [Ir(ppy)2Cl]2 (200.8 mg, 179.47 μmol) and the ligand L (80.0 mg, 200.8 μmol) were mixed and dispersed in an organic solvent (CH2Cl2 / CH3OH, v / v=2 / 1), and refluxed at 60°C for 24 hours under nitrogen protection. The reaction mixture was cooled, and the solvent was removed by rotary evaporation. N, N-dimethylformamide (DMF, 2 mL) and saturated potassium hexafluorophosphate solution (KPF6, 50 ml) were added to dissolve the solid sample, filtered, and washed with water to obtain a solid sample. The solid was purified by rapid column chromatography on a silica gel column (CH3CN / H2O, v / v=10 / 1), and the solvent was removed by evaporation under nitrogen. The product was a yellow solid, which was the cyclometalated Ir(III) complex.

[0066] Performance Testing

[0067] 1. Nuclear magnetic resonance and mass spectrometry analysis

[0068] The cyclometallated Ir(III) complex in Example 4 was analyzed by nuclear magnetic resonance spectrum using a Bruker Advance III 400 MHz spectrometer (Bruker, Germany) and by mass spectrometry using a Thermo Scientific LTQ linear ion trap mass spectrometer (Thermo Scientific, USA). The test results are as follows:

[0069] 1 H NMR (400 MHz, DMSO-d 6)δ10.54(s,1H),9.06(dd,J=8.6,1.4Hz,1H),8.85(dd,J=8.4,1.4Hz,1H),8 .64(s,1H),8.30–8.20(m,3H),8.14–8.02(m,3H),8.02–7.93(m,3H),7.93– 7.84(m,2H),7.47(d,J=5.7Hz,2H),7.32–7.13(m,6H),7.11–6.91(m,7H),6 .34–6.25(m,2H),2.82(t,J=7.0Hz,2H),2.72(t,J=7.4Hz,2H),1.24(s,2H). 13 C NMR (101MHz, DMSO) δ172.63,171.62,167.31,167.28,162.78,151.14,150.61,150.25,149. 96,149.59,147.00,144.52,144.47,144.25,139.95,139.18,138.66,135.10,134.64,131.6 9,131.32,130.71,129.09,128.78,127.69,127.53,126.95,126.54,125.55,124.39,122.86 ,120.46,119.73,40.77,36.25,35.66,31.90,31.24,30.66.ESI-MS(CH3OH)m / z:calculated for[M–PF6] + (C 46 H 38 IrN6O2 + )899.27, found 899.43.

[0070] 2. Cytotoxicity Evaluation

[0071] The CCK8 method was used to investigate the toxic effects of drug photodynamic therapy on oral squamous cell carcinoma cells. The cell lines were seeded in 96-well plates, with 5×10 3 The cells were placed in an incubator (37°C, 5% CO2) and cultured normally until the cells adhered to the wall. The original culture medium was then aspirated and different concentrations of drugs were added to the cells, 100 μL per well. Four parallel wells were set up, and blank culture medium and untreated cells were set up as control wells. After 24 hours of drug culture, the cells were illuminated with a 425nm laser source (20mWcm -2 ) for 15 min, and then continue to incubate for 24 h. 100 ul of CCK8 solution was added to each well and incubated at 37°C for 2 h. The absorbance value (OD) of each well was measured at 450 nm using a multifunctional microplate reader.

[0072] The cell survival rate was calculated as follows: cell survival rate % = (OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group).

[0073] The photodynamic activity of human tongue squamous cell carcinoma (CAL33, HSC6 and SCC1) and human oral mucosal precancerous cells (DOK) was evaluated in vitro using the cyclometalated Ir(III) complex of Example 4. The results are shown in Table 1 (unit: μM).

[0074] Table 1 Cytotoxicity test results

[0075]

[0076]

[0077] Ir1 (cyclometalated Ir(III) complex) has lower dark toxicity than cisplatin to all human tongue squamous cell carcinoma cells and has stronger photodynamic activity under photodynamic conditions. Among the cell lines tested, SCC1 cells are the most sensitive to PDT treatment. The phototoxicity index of Ir1 (PI: IC obtained under dark conditions and light conditions) is 50 The ratio of the values ​​of 1 and 2 is 120.43. The test results show that the cyclometallated Ir(III) complex has a strong PDT effect.

[0078] 3. Electron Spin Resonance Spectroscopy Analysis

[0079] The cyclometallated Ir(III) complex in Example 4 was subjected to electron spin resonance spectroscopy analysis using a JES-FA200 electron paramagnetic resonance spectrometer (JEOL, Japan). The test method was as follows: Ir (100 μM) and TEMP (10 mM) were mixed in PBS buffer and then illuminated with a 425 nm laser source (20 mW cm -2 ) for 3 min, and then immediately characterize and detect 1 O2 generation. Ir (100 μM) and DMPO (100 mM) were mixed in PBS buffer and then illuminated with a 425 nm laser source (20 mW cm -2 ) for 5 min and immediately characterize to detect the generation of ·OH. Figure 2 Experimental results: The test results show that Ir1 (cyclometalated Ir(III) complex) can produce 1 O2 and ·OH, indicating that the cyclometalated Ir(III) complex is a photosensitizer with both type I / II photodynamic effects.

[0080] 4. Organelle Localization Analysis

[0081] (1) Determine the fluorescence emission spectrum of the cyclometalated Ir(III) complex titrated with lecithin in Example 4. Specifically, a fluorescence spectrometer (FLS980 fluorescence spectrometer, Edinburgh, UK) is used to measure the fluorescence emission spectrum of Ir1 (10 μM) in Tris-HCl buffer under 405 nm excitation light. Wash and dry the cuvette, add the Ir1 (10 μM) solution to the cuvette, preheat the instrument, calibrate, and test the mode as spectral scanning and wavelength detection. The binding constant K is calculated using the Benesi–Hildebrand equation based on the fluorescence intensity of the sample at 600 nm. b .according to Figure 3 The experimental results show that at the emission maximum, the fluorescence emission intensity of Ir1 increased by 3 times, and the binding constant was 1.6×10 4 M -1 , indicating that it can effectively bind to lecithin.

[0082] (2) The colocalization of the cyclometallated Ir(III) complex in Example 4 with the endoplasmic reticulum was determined by incubating SCC1 cells with Ir1 (10 μM) for 30 min and applying light (425 nm, 20 mW cm -2 ) for 15 min, add endoplasmic reticulum red probe (ERTR, 1 μM) and incubate for 15 min, then take pictures using a confocal microscope (LSM880 with fast AiryScan, ZEISS, Germany), and analyze the results using Zen software, scale: 10 μm. Figure 4 The experimental results showed that after Ir1 was co-incubated with cells, the fluorescence of Ir1 and ERTR in the cells basically coincided, and the Pearson correlation coefficient (PCC) was 0.83. The test results showed that Ir1 could effectively target the endoplasmic reticulum.

[0083] 5. Analysis of Cellular ROS Production Ability

[0084] The ability of the cyclometalated Ir(III) complex in Example 4 to generate ROS was determined by flow cytometry. The specific method was as follows: SCC1 cells with good growth status were taken, routinely rinsed, digested and counted, and 2×10 5Cells were seeded at a density of cells / well in a 6-well plate and placed in an incubator (37°C, 5% CO2) for normal culture until the cells adhered to the wall. A certain concentration of Ir1 (0.2μM, 0.4μM, 0.8μM) was added, and 3 replicate wells were set up in each group. The plates were placed in a 37°C incubator and cultured in the dark for 24 hours. Irradiate with a 425nm laser for 15 minutes, incubate in the dark for 30 minutes, then add 10μM DCFH DA dye and incubate at 37°C in the dark for 20 minutes. Wash with PBS, digest the cells with EDTA-free trypsin for 2 minutes, add culture medium to terminate digestion, collect the cell suspension, centrifuge at 1000rpm for 3 minutes, discard the supernatant, suspend the cells, centrifuge again, discard the supernatant, add 500μL PBS to each tube to suspend the cells, and then apply to the flow cytometer. According to Figure 5 The experimental results showed that Ir1 (0.2μM, 0.4μM, 0.8μM) could induce ROS generation through PDT. At a concentration of 0.8μM, Ir1 increased the cell DCF fluorescence by about 11.7 times. The test results showed that it could effectively generate reactive oxygen species.

[0085] 6. Analysis of anti-tumor effects

[0086] (1) Analysis of endoplasmic reticulum stress effects

[0087] 1) Analyze the expression levels of ER stress-related proteins in cells by Western Blot.

[0088] The specific method is as follows: SCC1 cells were cultured with Ir1 (0.2, 0.4, 0.8 μM) in a 37°C incubator for 24 hours, irradiated with a 425 nm laser for 15 minutes, and the cells were collected 3 hours later. The collected cells were lysed on ice for 30 minutes with RIPA buffer containing 1% phosphatase inhibitors and 1% protease inhibitors. The protein concentration was quantified with BCA, and the sample was transferred to the membrane for electrophoresis and blocked, and then incubated with the primary antibody at 4°C overnight. After washing, the secondary antibody was incubated. The gel imager was used for color exposure to collect images, and the ImageJ software was used for analysis.

[0089] according to Figure 6 The experimental results showed that Ir1 (0.2μM, 0.4μM, 0.8μM) could induce the phosphorylation level of eIF2α, a key protein of endoplasmic reticulum stress, and upregulate the expression level of CHOP through PDT. The test results showed that it could induce the generation of endoplasmic reticulum stress through the typical pathway dependent on eIF2α.

[0090] 2) Transmission electron microscopy was used to observe the changes in cell morphology after Ir-PDT treatment.

[0091] The specific method is as follows: SCC1 cells with good growth status were inoculated into a 6 cm culture dish and cultured in a 37°C incubator to allow the cells to adhere to the wall. SCC1 cells were cultured with Ir1 (0, 0.8 μM) in a 37°C incubator for 24 hours, and then irradiated with a 425 nm laser for 15 minutes. After 3 hours, the cells were collected by trypsin digestion. The collected cells were washed 3 times with pre-cooled PBS and centrifuged at 1000 rpm for 5 minutes. The obtained cell mass was fixed with glutaraldehyde at 4°C overnight. After the sample was stained with osmic acid, the cell morphology was observed using a transmission electron microscope (TEM, Hitachi HT7800, Japan). Scale bar: 5 μm and 2 μm.

[0092] according to Figure 7 The experimental results showed that compared with the control group (no drug, light), the cells treated with Ir-PDT showed typical ultrastructural features of damage, including endoplasmic reticulum swelling and vacuolization. The test results further confirmed that Ir-PDT treatment can effectively induce the generation of endoplasmic reticulum stress.

[0093] (2) Analysis of immunogenic death effect

[0094] 1) In order to verify that Ir-PDT treatment increases the expression of CRT on the cell surface, SCC1 cells with good growth status were taken and 2×10 5 Cells were seeded in a 6-well plate at a density of 100 cells / well and cultured in a 37°C incubator to allow the cells to adhere to the wall. After washing with PBS, three concentrations of Ir, 0.2μM, 0.4μM, and 0.8μM, were added. Three replicate wells were set up in each group and placed in a 37°C incubator in the dark for 24 hours. 425nm laser irradiation for 15 minutes, after incubation for 3 hours, the cells were collected, washed with PBS 3 times, and fixed with 4% PFA at room temperature for 15 minutes. Then, Calreticulin (D3E6) was added. Rabbit mAb (Alexa The cells were incubated with 488Conjugate at 4°C for 12 h and analyzed by flow cytometry.

[0095] according to Figure 8 The experimental results showed that the expression of CRT on the cell surface increased after Ir-PDT treatment, and the expression increased with the increase of drug concentration.

[0096] 2) In order to verify that Ir-PDT treatment reduces intracellular HMGB1 expression, SCC1 cells with good growth status were taken and 2×10 5Cells were inoculated in 6-well plates at a density of 100 cells / well and cultured in a 37°C incubator to allow the cells to adhere to the wall. After washing with PBS, three concentrations of Ir were added: 0.2μM, 0.4μM, and 0.8μM. Three replicate wells were set up in each group and cultured in a 37°C incubator in the dark for 24 hours. After irradiation with 425nm laser for 15 minutes and incubation for 3 hours, the cells were collected, washed with PBS 3 times, and fixed with 4% PFA at room temperature for 15 minutes. Subsequently, 0.1% Triton X-100 was used to permeabilize the cells at room temperature for 15 minutes, and then incubated with PE anti-HMGB1 Antibody at 4°C for 12 hours and analyzed by flow cytometry.

[0097] according to Fig. 9 Experimental results: Flow cytometry showed that HMGB1 expression decreased after Ir-PDT treatment, and the expression decreased with the increase of drug concentration.

[0098] 3) In order to verify that Ir-PDT treatment promotes cellular ATP release, SCC1 cells with good growth status were taken and cultured at 6×10 3 Cells were inoculated into 24-well plates at a density of 100 cells / well and cultured in a 37°C incubator to allow the cells to adhere to the wall. After washing with PBS, three concentrations of Ir, 0.2μM, 0.4μM, and 0.8μM, were added. Three replicate wells were set up in each group and placed in a 37°C incubator in the dark for 24 hours. 425nm laser irradiation was performed for 15 minutes. After incubation for 3 hours, the supernatant was collected. The supernatant was then transferred to a black-walled, transparent-bottom 96-well plate, ATP detection reagent was added, and ATP levels were detected using chemiluminescence on an ELISA reader.

[0099] according to Fig.10 Experimental results: Chemiluminescence detection results showed that ATP release increased after Ir-PDT treatment, and the release increased with the increase of drug concentration.

[0100] 4) Analyze the PD-L1 expression level in cells by RT-qPCR and Western Blot.

[0101] The specific method of RT-qPCR detection is as follows: SCC1 cells were cultured with Ir1 (0.2, 0.4, 0.8 μM) in a 37°C incubator for 24 h, irradiated with 425 nm laser for 15 min, and incubated in the incubator for 3 h. Total RNA of the sample was extracted according to the instructions of the kit, and the RT-qPCR reverse transcription system was prepared. The reverse transcription instrument was placed in reverse transcription, and the RT-qPCR reaction system was prepared and placed in the LightCycler 96 fluorescence real-time quantitative PCR instrument to start the reaction. The target gene and the internal reference gene were compared according to 2 -ΔΔCT Methods Relative gene expression levels were calculated and normalized using GAPDH.

[0102] The specific method of Western Blot detection is as follows: SCC1 cells were cultured with Ir1 (0.2, 0.4, 0.8 μM) in a 37°C incubator for 24 hours, irradiated with a 425 nm laser for 15 minutes, and the cells were collected 3 hours later. The collected cells were lysed on ice for 30 minutes with RIPA buffer containing 1% phosphatase inhibitors and 1% protease inhibitors. The protein concentration was quantified with BCA, and the sample was transferred to the membrane for electrophoresis and blocked, and then incubated with the primary antibody at 4°C overnight. After washing, the secondary antibody was incubated. The gel imager was used for color exposure to collect images, and the images were analyzed using ImageJ software.

[0103] according to Fig.11 The experimental results showed that Ir1 (0.2μM, 0.4μM, 0.8μM) can induce upregulation of PD-L1 expression levels after PDT. The above test results further confirmed that Ir-PDT treatment can effectively induce the immunogenic death of tumor cells and upregulate the expression of PD-L1.

[0104] (3) Evaluation of the anti-tumor effect of Ir-PDT using the CDX model

[0105] Six-week-old C57BL / 6 male mice were housed in the SPF barrier system of Sun Yat-sen University, and feed and drinking water were sterilized in the animal room and provided to the animals ad libitum.

[0106] 100 μL (1×10 6 The CDX model was established by using a live cell suspension of SCC7 (squamous cancer cell line) and combined with photodynamic therapy. 3 The mice were randomly divided into the following four groups, with 6 mice in each group: (1) normal saline (50 μL); (2) Ir1 (50 μL, 5 mg / kg) + light; (3) PD-L1 inhibitor (BMS1, 50 μL, 2.5 mg / kg); (4) Ir1 + light + PD-L1 inhibitor. Ir1 was injected intratumorally once a week for a total of 2 times. After administration, the tumor was treated with 425 nm laser (20 mW cm -2 ,30min) for combined photodynamic therapy. PD-L1 inhibitors were injected intraperitoneally once every 3 days for a total of 6 times. The tumor volume and body weight of mice were recorded every other day from the day of administration. The mice were killed 17 days after treatment, and the tumors were removed and weighed. Part of the tumor tissue was fixed with 10% buffered formalin, embedded in paraffin, and stained with H&E and IHC for Ki67 and PD-L1. Scale: 60μm (H&E), 20μm (IHC). Part of the tumor tissue was taken for flow cytometry and cytokine detection. The main organs of the rats, such as heart, liver, spleen, lung, and kidney, were taken and embedded in paraffin for histopathological examination. Scale: 100μm.

[0107] The experimental results are shown in Fig.12 , (a) is the tumor volume growth curve during the treatment; (b) is the tumor tissue diagram after the end of treatment; (c) is the tumor tissue weight analysis after the end of treatment; (d) is the HE and IHC staining analysis of the tumor tissue after the end of treatment; (e) is the HE staining analysis of the organ tissue after the end of treatment; (f) is the mouse weight curve during the treatment; (g) is the flow cytometry analysis of the synergistic enhancement of the anti-tumor immune response during the treatment; (h) is the ELISA analysis of the expression of anti-tumor immune-related cytokines in the tumor tissue after the end of treatment.

[0108] According to the tumor volume growth curve, tumor tissue macrograph and tumor tissue weight bar graph, it can be seen that the Ir1+light irradiation group and PD-L1 inhibitor respectively have a significant inhibitory effect on tumor growth, and their combined effect more significantly causes tumor regression.

[0109] HE results showed that the tumor tissue in the Ir1-PDT and PD-L1 inhibitor combined treatment group had severe necrosis, obvious cavitation, a large number of cell nuclei missing, and obvious nuclear condensation, indicating that this treatment regimen has an effective anti-tumor killing effect. IHC results showed that the Ki67 positivity rate in the Ir1-PDT group and the combined treatment group with PD-L1 inhibitor was significantly reduced, indicating that this treatment regimen has a strong inhibitory effect on tumor cell proliferation. In addition, in the Ir-PDT group, the PD-L1 positivity rate was significantly upregulated, and in the Ir-PDT combined treatment group with PD-L1 inhibitor, the high expression of PD-L1 mediated by Ir1-PDT was reversed by PD-L1 inhibitor, thereby alleviating immunosuppression.

[0110] Flow cytometry results showed that the tumor cell CRT in the Ir1-PDT and PD-L1 inhibitor combined treatment group + Increased proportion of MHC II on dendritic cells + and CD80 + CD86 + Significantly upregulated, tumor-specific CD4 + and CD8 + T cell infiltration levels were upregulated, and Ir1-PDT induced CD8 + The increased expression of PD-1 on the surface of T cells was reversed by PD-L1 inhibitors, indicating that this treatment regimen can induce immunogenic death, promote dendritic cell maturation, and have the effect of synergistically enhancing anti-tumor adaptive immunity.

[0111] ELISA results showed that the levels of TNF-α and IFN-γ in tumor tissues were the highest in the Ir1-PDT and PD-L1 inhibitor combined treatment group. Compared with the control group, the immunosuppressive inflammatory cytokine IL-6 was significantly downregulated after combined treatment, further confirming its tumor killing ability in the tumor microenvironment and enhancing the anti-tumor immune response.

[0112] At the same time, no obvious toxic side effects were found in the HE sections of the main organs of mice, and the weight of mice in all groups did not change significantly during the treatment, and there was no sharp weight loss or gain in a short period of time, indicating that the drug administration is relatively safe.

[0113] The above test results confirm that the combined treatment of Ir1-PDT and PD-L1 inhibitors has a strong tumor inhibitory effect and can alleviate the immunosuppressive tumor microenvironment. This combined treatment can transform "immune-cold" tumors into "immune-hot" tumors and synergistically enhance anti-tumor adaptive immunity. It is feasible and has high biosafety.

[0114] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. 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 claims of the present invention.

Claims

1. A cyclometalated Ir(III) complex, characterized in that The structural formula of the cyclometalated Ir(III) complex is shown in formula (I):

2. A method for preparing the cyclometallated Ir(III) complex according to claim 1, characterized in that: The following steps are involved: The bridged iridium precursor [Ir(ppy)2Cl]2 and the ligand L are mixed and dispersed in an organic solvent, reacted at 50-70°C for 16-32h under the protection of an inert gas, and purified to obtain a cyclometalated Ir(III) complex; Wherein, the structural formula of the ligand L is shown in formula (II):

3. The preparation method according to claim 2, characterized in that: The molar ratio of the bridging iridium precursor [Ir(ppy)2Cl]2 to the ligand L is 1:(2.0-2.5).

4. The preparation method according to claim 2, characterized in that: The organic solvent is a mixture of CH2Cl2 and CH3OH in a volume ratio of (1-3):

1.

5. The preparation method according to claim 2, characterized in that: The ligand L is prepared by the following preparation method: The reaction monomer I, O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, N,N′-diisopropylethylamine and 1,10-phenanthroline-5-amino were uniformly mixed, reacted at 40-50° C. for 6-10 hours, and purified to obtain the ligand L; Wherein, the structural formula of the reaction monomer I is shown in formula (III):

6. The preparation method according to claim 5, characterized in that: The molar ratio of the reaction monomer I to 1,10-phenanthroline-5-amino is 1:(1.0-1.2).

7. The preparation method according to claim 5, characterized in that: The reactive monomer I is prepared by the following preparation method: β-phenylethylamine and succinic anhydride are dissolved in an organic solvent and reacted at 40-50° C. for 2-8 hours to obtain reaction monomer I.

8. Use of the cyclometallated Ir(III) complex according to claim 1 in the preparation of antitumor materials or antitumor drugs.

9. The use according to claim 8, wherein the anti-tumor material is an anti-oral squamous cell carcinoma material, and the anti-tumor drug is an anti-oral squamous cell carcinoma drug.

10. An anti-tumor photodynamic therapy agent, characterized in that: The anti-tumor photodynamic therapy agent comprises the cyclometalated Ir(III) complex according to claim 1.

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