An iridium complex for dynamic detection of mitochondrial viscosity and tumor differentiation, and a preparation method and use thereof

Abstract

The application relates to the field of biological medicine, and discloses an iridium complex for dynamic detection of mitochondrial viscosity and tumor differentiation, a preparation method and application thereof, and a structure formula of the iridium complex is as follows: The iridium complex Ir-visc in the application is based on a double-locking targeting strategy of electrostatic adsorption and probe-protein docking, does not depend on MMP, and stably anchors mitochondria in cells; the iridium complex exhibits excellent high-viscosity-activated one-photon (OP) and two-photon (TP) phosphorescence characteristics, is not disturbed by other factors in a living body, can be used for monitoring viscosity changes in a complex biological system; can be used as a mitochondrial phosphorescence probe to differentiate tumor cells and normal cells, and monitor changes of mitochondrial viscosity of cells under the stimulation of starvation and an antifungal drug nystatin; and not only has strong tumor tissue recognition capability, but also has deep tissue penetration and in-vivo imaging capability.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, and discloses an iridium complex material for dynamic detection of mitochondrial viscosity and tumor differentiation and a preparation method thereof, in particular to an iridium complex with high quantum yield under viscosity activation, which can be used for selectively illuminating cancer cells and monitoring mitochondrial viscosity, and a preparation method and application thereof. BACKGROUND

[0002] Fluorescence imaging technology has the advantages of non-invasiveness, ultra-sensitivity, high selectivity and easy use, and shows great potential and application prospect in the field of cancer diagnosis. Viscosity is an important indicator of the subcellular microenvironment, and plays a key role in regulating the transport of nutrients and metabolic waste, promoting signal transduction and other biological processes. Abnormal changes in viscosity are closely related to malignant tumors. The characteristics of malignant tumors are uncontrolled growth of cancer cells and accumulation of high-viscosity metabolic products, especially in mitochondria. Therefore, mitochondrial viscosity may be a potential target for designing activatable probes to identify cancer cells. With mitochondrial viscosity as the link, the microenvironment around early tumor tissues is perceived, which is crucial for tracking tumor occurrence and detecting tumors. However, the high dependence of most viscosity-activated mitochondrial probes on mitochondrial membrane potential (MMP), as well as the short luminescence lifetime and poor tissue penetration depth seriously hinder their wide application. Therefore, it is crucial to develop mitochondrial viscosity-activated probes independent of MMP, accurately and reliably locate and quantitatively monitor mitochondrial viscosity in the fields of biological research, cancer diagnosis and drug development.

[0003] The luminescent ring metalated iridium (III) complex [Ir(C^N)2(N^N)]+ with octahedral configuration is widely used in the fields of biological imaging and biosensing due to its high quantum yield, large Stokes shift, long luminescence lifetime, good light stability and other advantages. In addition, through ligand modification, the iridium complex can easily obtain excellent two-photon absorption (TPA) performance, thereby having the characteristics of tissue penetration, low photobleaching, low phototoxicity and low photo-damage, and weak autofluorescence. SUMMARY

[0004] The application aims to provide an iridium complex material for dynamic detection of mitochondrial viscosity and tumor differentiation and a preparation method thereof, in particular to an iridium complex with high quantum yield under viscosity activation, which can be used for selectively illuminating mitochondria in deep tissue cancer cells. To achieve the above-mentioned purpose, the application provides the following technical solutions.

[0005] The design concept of the present application includes: the present application uses a double-locking mitochondria targeting strategy, and reasonably designs an iridium complex (Ir-visc) with viscosity-activated TPA absorption activity to cope with the above challenges. First, a benzothiazole derivative is used as a C^N ligand, and the structure of the benzothiazole platform is modified to adjust the biological activity and enhance the binding capacity of the probe protein molecule; second, an electron-rich thiophene ring is used as a molecular rotor, and a single bond is used to connect the pyrazole ring to form an N^N ligand, so as to adjust the charge distribution and viscosity-activated properties of the iridium complex Ir-visc. Finally, the iridium complex Ir-visc carries a positive charge, and can drive the accumulation of mitochondria through the negative membrane potential. In addition, the synergistic effect of the multi-aromatic ring and the positive charge enables Ir-visc to bind to mitochondrial membrane proteins through the formation of non-covalent bonds (such as hydrogen bonds, ionic bonds and van der Waals forces).

[0006] In a first aspect, the present application provides an iridium complex, and the structure of the complex is as follows:

[0007]

[0008] In a second aspect, the present application also provides a preparation method of an iridium complex, comprising the following steps:

[0009] C ^ Synthesis of C^N ligand L1: 3,4-diethoxybenzaldehyde and o-aminothiophenol were dissolved in ethanol, and after heating and refluxing, a white filamentous product was obtained. After vacuum filtration and drying, C ^ N ligand L1 was obtained.

[0010] Synthesis of intermediate iridium chloride bridge M: under the protection of nitrogen and in the dark, a Schlenk flask was added with the above C ^ N ligand and iridium chloride trihydrate, and then ethylene glycol monoethyl ether and water were added as solvents. After heating and refluxing, the solution was cooled to room temperature, and orange solid was precipitated. After vacuum filtration, the intermediate iridium chloride bridge M was obtained.

[0011] N ^ Synthesis of N^N ligand L2: under the protection of nitrogen, sodium hydride in tetrahydrofuran was added to a three-necked flask, and the flask was slowly cooled to room temperature in an ice bath. Pyrazole in tetrahydrofuran was slowly added to form a light yellow solution, and then dichlorosulfoxide was slowly added to obtain a yellow-white suspension. After stirring at room temperature, 2-thiophene aldehyde and anhydrous cobalt chloride were added for heating and refluxing. After cooling to room temperature, water was added, and then dichloromethane was used for extraction. Anhydrous magnesium sulfate was dried overnight, and then rotary evaporation and column chromatography were performed to obtain N ^ N ligand L2.

[0012] Synthesis of complex Ir-visc: under the protection of nitrogen and in the dark, a Schlenk flask was added with the intermediate iridium chloride bridge M, N ^The yellow solid is obtained by stopping the reaction and cooling after refluxing the reaction with a mixed solution of N ligand L2 and ammonium hexafluorophosphate, methanol and dichloromethane as solvent.

[0013] Further, the molar ratio of 3,4-diethoxybenzaldehyde to 2-aminothiophenol is 1:1-1.2.

[0014] Further, the molar ratio of C ^ The molar ratio of N ligand L1 to iridium trichloride trihydrate is 2-2.5:1; the volume ratio of ethylene glycol monoethyl ether to water in the mixed solvent is 3:1.

[0015] Further, the molar ratio of pyrazole, dichlorosulfoxide and 2-thiophene aldehyde is 2:1:0.2-0.5; the molar ratio of sodium hydride to pyrazole is 1:1; the molar ratio of anhydrous cobalt chloride to 2-thiophene aldehyde is 1:3-3.5.

[0016] Further, the preparation steps of the intermediate iridium chloride bridge M, N ^ The molar ratio of N ligand L2 to ammonium hexafluorophosphate is 1:2-3:2-4; the volume ratio of methanol to dichloromethane in the mixed solvent is 1:1.

[0017] Further, the conditions of heating and refluxing reaction are as follows:

[0018] C ^ In the preparation steps of N ligand L1: the temperature is 70-80℃, and the time is 6-8h;

[0019] In the preparation steps of the intermediate iridium chloride bridge M: the temperature is 110-120℃, and the time is 24-30h;

[0020] N ^ In the preparation steps of N ligand L2: the temperature is 70-80℃, and the time is 10-12h;

[0021] In the preparation steps of the iridium complex: the temperature is 75-80℃, and the time is 12-16h.

[0022] In a third aspect, the application also provides a use of the iridium complex for preparing a composition, a probe or a kit for detecting mitochondrial viscosity and / or distinguishing tumors.

[0023] Technical effects and advantages of the application:

[0024] 1. The iridium complex Ir-visc is based on the double-locking targeting strategy of electrostatic adsorption and probe-protein docking, and does not depend on MMP, and can stably anchor mitochondria in cells.

[0025] 2. The iridium complex Ir-visc exhibits excellent high-viscosity activated one-photon (OP) and two-photon (TP) phosphorescence properties, and is not interfered by other factors in the organism. It can be used to monitor changes in viscosity in complex biological systems.

[0026] 3. The iridium complex Ir-visc has been successfully used as a mitochondrial phosphorescence probe to distinguish tumor cells from normal cells through OP and TP phosphorescence imaging, and to monitor changes in cell mitochondrial viscosity under starvation and stimulation with the antifungal drug nystatin.

[0027] 4. The iridium complex Ir-visc not only has strong tumor tissue recognition capabilities, but also has deep tissue penetration and in vivo imaging capabilities, providing a multi-level, multi-dimensional visualization tool for cancer diagnosis and cancer-related clinical research.

[0028] Other features and advantages of the present invention will be described in the following description, and in part will become apparent from the description, or will be understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures pointed out in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1a is a time-of-flight mass spectrum of the iridium complex Ir-visc of the present invention;

[0031] Figure 1b is the hydrogen nuclear magnetic resonance spectrum of the iridium complex Ir-visc of the present invention;

[0032] Figure 1c is the carbon NMR spectrum of the iridium complex Ir-visc of the present invention;

[0033] Figure 2 a is a phosphorescence spectrum of the iridium complex Ir-visc of the present invention in different solvents and glycerol;

[0034] Figure 2 b is the phosphorescence spectrum of the iridium complex Ir-visc of the present invention in glycerol and methanol solvents at different ratios;

[0035] Figure 2 c is the iridium complex Ir-visc of the present invention at different viscosities (Logη) and phosphorescence intensity (LogI575nm ) of the present application;

[0036] Figure 2 d is the phosphorescence emission spectrum of the iridium complex Ir-visc of the present application in different pH and glycerol solutions;

[0037] Figure 2 e is the selectivity of the iridium complex Ir-visc of the present application to various biologically relevant species;

[0038] Figure 2 f is the TPA cross-section (λ ex = 680-1000 nm, 500 mW) of the iridium complex Ir-visc of the present application in methanol and glycerol (80%);

[0039] Figure 3 a is the confocal image of the iridium complex Ir-visc of the present application co-stained with mitochondrial dye (Mito-Lite Green) and nuclear dye (Hochest 33342) in HepG2 cells;

[0040] Figure 3 b is the confocal image of the iridium complex Ir-visc of the present application after co-incubation with HepG2 cells under one-photon (OP) and two-photon (TP) excitation;

[0041] Figure 4 a is the confocal image of the iridium complex Ir-visc of the present application for monitoring the change of mitochondrial phosphorescence in HepG2 cells before and after CCCP treatment;

[0042] Figure 4 b is the interaction diagram between the iridium complex Ir-visc of the present application and the adjacent residues of protein (OPA1);

[0043] Figure 5 a is the confocal image of the iridium complex Ir-visc of the present application after incubation with tumor cells (HeLa cells and HepG2 cells) and normal cells (HUVEC cells and HL-7702 cells), respectively;

[0044] Figure 5 b is the quantification image of the relative phosphorescence intensity of the iridium complex Ir-visc of the present application after incubation with tumor cells (HeLa cells and HepG2 cells) and normal cells (HUVEC cells and HL-7702 cells), respectively;

[0045] Figure 5 c is the confocal image of the iridium complex Ir-visc of the present application after treatment of HepG2 cells with Nystatin and serum-free medium (HBSS), respectively;

[0046] Figure 5 d is the relative phosphorescence intensity quantification image of the iridium complex Ir-visc of the application after treating HepG2 cells with Nystatin and HBSS respectively;

[0047] Figure 6 a is the OP and TP confocal image of the iridium complex Ir-visc of the application to the tumor tissue and normal tissue area of LLC tumor-bearing mice;

[0048] Figure 6 b is the image of the iridium complex Ir-visc of the application to distinguish tumor tissue and normal tissue by OP and TP imaging means;

[0049] Figure 6 c is the OP and TP three-dimensional phosphorescence depth image of the iridium complex Ir-visc of the application after treating tumor tissue. DETAILED DESCRIPTION

[0050] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0051] The flowchart shown in the drawings is only an exemplary illustration, and it is not necessary to include all the steps. For example, some steps can be further decomposed, and some steps can be combined or partially combined, so that the actual execution order can be changed according to the actual situation.

[0052] The terms "first", "second", and the like in the specification and claims of the present application and the above-mentioned drawings are used to distinguish similar objects, and do not necessarily describe a specific order or sequence. It should be understood that the data thus used can be interchanged under appropriate circumstances, so that the embodiments of the application described herein can be implemented in an order other than those illustrated or described herein.

[0053] Example 1: Preparation of an iridium complex

[0054] Step S1, synthesis of C^N ligand L1: take 1.94 g (10.0 mmol) of 3,4-diethoxybenzaldehyde and 1.25 g (10.0 mmol) of 2-aminothiophenol, add to a 100 mL round-bottom flask, dissolve with an appropriate amount of ethanol (dissolve immediately), reflux at 75°C for 6 h, cool and precipitate white needle-shaped product, dry to obtain 2.1 g of C^N ligand L1, yield 70%. m / z: [M+H + ] +:300.1045g / mol(calculated:300.1052g / mol). 1 H-NMR (400MHz, DMSO) δ8.10 (2H, d, J = 7.9 Hz), 8.02 (2H, d, J = 8.0 Hz), 7.64 (2H, d, J = 2.0 Hz), 7.58 (2H, dd, J = 8. 3,2.1Hz),7.54-7.50(2H,m),7.44-7.41(2H,m),7.11(2H,d,J=8.4),4.21-4.04(8H,m),1.42-1.33(12H,m). 13 C-NMR (101MHz, DMSO) δ166.65,153.02,150.32,147.81,133.68,125.91,124.80,124.33,121.87,121.56,120.10,112.40,110.16,63.13,14.01.

[0055]

[0056] Step S2, synthesis of the intermediate iridium chloride bridge M: Under nitrogen and light protection, 0.65 g (2.2 mmol) of L1 and 0.35 g (1.0 mmol) of iridium (III) chloride trihydrate were added to a 50 mL Shrek bottle, 30 mL of ethylene glycol monoethyl ether and 5 mL of water were added as a mixed solvent, the temperature was raised to 110 ° C and refluxed for 24 h. After cooling, an orange solid precipitated to obtain 0.61 g of the intermediate iridium chloride bridge M with a yield of 74%.

[0057]

[0058] Step S3, Synthesis of N^N Ligand L2: Under nitrogen protection, 1.16 g (48 mmol) of NaH was added to a three-necked flask containing 50 mL of refined THF. The mixture was slowly cooled to 0°C in an ice bath. 3.27 g (48 mol) of pyrazole in THF (15 mL) was slowly added over 15 minutes and stirred for 30 minutes to form a light yellow solution. 2.91 g (24 mmol) of SOCl2 was then slowly added to obtain a yellow-white suspension. The mixture was removed from the ice bath and reacted at room temperature for 40 minutes. 0.54 g (4.8 mmol) of 2-thiophenealdehyde and 0.19 g (1.4 mmol) of anhydrous cobalt chloride were then added to the above system. The mixture was refluxed for 10 hours, cooled to room temperature, and 40 mL of water was added. The mixture was stirred for 10 minutes, extracted with dichloromethane, dried over anhydrous magnesium sulfate overnight, and then dried by spin drying. The mixture was purified by column chromatography using petroleum ether:ethyl acetate (5:1) to obtain 0.58 g of a white solid with a yield of 53%. MS:m / z:[M+Na] +:253.0519g / mol(calculated:253.0518g / mol). 1 H-NMR (400MHz, DMSO) δ8.32 (1H, s), 7.94 (2H, d, J = 2.3Hz), 7.64 (1H, dd, J = 5.0, 1.3Hz), 7 .59(2H,d,J=1.5Hz),7.08-7.06(1H,m),7.04(1H,dd,J=5.0,3.6Hz),6.34-6.33(2H,m). 13 C-NMR (101MHz, DMSO) δ139.51,137.78,129.32,127.65,127.48,126.03,105.61,71.82.

[0059]

[0060] Step S4, synthesis of complex Ir-visc: under nitrogen and light-proof conditions, 50 mL of a solution of methanol and dichloromethane (1:1) was used as solvent, 0.83 g (0.50 mmol) of the intermediate iridium chloride bridge M, 0.29 g (1.28 mmol) of N ^ N ligand L2 and 0.33 g (2.00 mmol) NH4PF6 were cooled to room temperature and refluxed at 75°C for 12 h to precipitate 0.68 g of a yellow solid (Ir-visc). Yield: 58%. MS: m / z: [M-PF6 - ] + :1019.18g / mol(calculated:1019.21g / mol). 1H-NMR (400MHz, DMSO) δ8.59(2H,dd,J=17.1,2.2Hz),8.12(1H,d,J=7.9Hz),8.00(1H,d,J=7.9Hz),7.42(1H,s),7.34(2H,dd,J= 15.2,8.0Hz),7.27-7.17(1H,m),7.09(2H,dd,J=11.3,4.3Hz),6.99-6.89(3H,m),6.82(1H,dd,J=8.3,5.9Hz),6.58(2H,dt,J=1 8.3,12.2Hz),6.00(1H,dd,J=4.9,3.7),5.71(1H,s),5.60(1H,d,J=3.3Hz),5.46(2H,d,J=9.4Hz),5.11(1H,t,J=9.7Hz),4.07 -3.91(2H,m),3.45(2H,dt,J=14.0,7.1Hz),3.18-3.01(2H,m),1.36-1.17(6H,m),0.84(3H,t,J=6.9Hz),0.69(3H,t,J=6.9Hz). 13 C-NMR (151MHz, CDCl3) δ164.36,164.00,141.73,139.94,137.42,136.72,136.47,136.10,132.46,129.28,126.03,125. 48,123.94,122.30,121.31,120.42,118.34,115.17,112.79,108.96,107.24,78.82,72.12,71.01,70.74,31.97,31.29.

[0061]

[0062] Example 2: Biological studies of target molecules

[0063] In order to explore the spectroscopic behavior of the iridium complex Ir-visc in different polar solvents and glycerol, we selected dimethyl sulfoxide (DMSO), dichloromethane (DCM), acetonitrile (CH3CN), ethyl acetate (EA), water (H2O), methanol (MeOH), tetrahydrofuran (THF), ethanol (EtOH) and glycerol (Gly). Figure 2 a is the emission spectrum of the iridium complex Ir-visc of the present invention in solvents of different polarities and glycerol, as shown in Figure 2As shown in Figure a, in solvents of different polarities, the free rotation of single bonds in the complex induces non-radiative energy transitions and consumes the energy of the excited state. Therefore, the phosphorescence emission of iridium complexes in different polarity solutions is low. In glycerol, the high viscosity environment restricts the free rotation of Ir-visc, and an obvious emission peak enhancement is observed at 575 nm.

[0064] In order to accurately verify the relationship between the phosphorescence intensity of the iridium complex Ir-visc and viscosity, a solvent with a gradient increase of glycerol and methanol ratio of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% was used as a viscosity detection system. Figure 2 b is the phosphorescence emission spectrum of the iridium complex Ir-visc of the present invention in different ratios of glycerol / methanol (0%-100%), as shown in FIG. Figure 2 As shown in b, with the increase of glycerol ratio, the emission intensity of Ir-visc at 575nm gradually increased, showing a higher viscosity sensitivity. More importantly, Figure 2 c is the phosphorescence intensity (LogI) of the iridium complex Ir-visc at different viscosities (Logη) and 575nm 575nm ) linear correlation diagram, according to log I 572nm =0.6766logη+1.800 Equation, in the range of 0.55 to 944 cP, log I 572nm There is a good linear relationship between Ir-visc and logη, with a correlation coefficient as high as 0.978, indicating that Ir-visc has a high sensitivity in detecting the viscosity of the cell microenvironment.

[0065] To demonstrate that the generation of Ir-visc emission signals is solely related to changes in viscosity, different solvent systems were studied. Figure 2 d is the phosphorescence emission spectrum of the iridium complex Ir-visc of the present invention in different pH and glycerol solutions, as shown in FIG. Figure 2 As shown in Figure d, the phosphorescence emission spectra of Ir-visc are hardly affected in different acidic and alkaline environments.

[0066] Furthermore, to prove the specificity of Ir-visc response to viscosity, Figure 2 e is the selectivity spectrum of the iridium complex Ir-visc of the present invention to various biologically relevant species, such as Figure 2 As shown in Figure e, different interference systems, including various anions, cations and amino acids, were added to the aqueous solution of the iridium complex Ir-visc, and the emission spectrum did not change significantly, indicating that the iridium complex Ir-visc can be used to monitor changes in viscosity in complex biological systems.

[0067] The strong push-pull electron effect within the Ir-visc molecule may enable it to acquire TPA activity. Figure 2 f is the TPA cross section (λ) of the iridium complex Ir-visc of the present invention in methanol and glycerol (80%) ex =680-1000nm,500mW), such as Figure 2 Figure f shows the TP absorption cross sections of the iridium complex Ir-visc in methanol and glycerol under excitation wavelengths of 680 to 1000 nm, a pulse duration of 140 fs, and a power of 500 mW. Under 840 nm excitation, Ir-visc exhibits an optimal TPA cross section, with the TPA cross section in glycerol (76.33 GM) being nearly 4.2 times higher than that in methanol (18.23 GM), indicating its potential as an excellent tool for studying mitochondrial-related pathological processes in deep tissues.

[0068] In order to verify the targeting of iridium complex Ir-visc to mitochondria in the cellular environment, a mitochondrial dye without spectral cross-talk (Mito-Lite Green, λ ex =490nm,λ em =500-550nm), nuclear dye (Hoechst33342, λ ex =346nm,λ em =430-470 nm) and Ir-visc were incubated with HepG2 cells for co-localization experiments. Figure 3 a is a confocal image of HepG2 cells co-stained with the iridium complex Ir-visc of the present invention, a mitochondrial dye (Mito-Lite Green), and a nuclear dye (Hochest 33342). Figure 3 As shown in a, the colocalization coefficient of the iridium complex Ir-visc with Mito-Lite Green (green) in HepG2 cells is as high as 0.918, which is significantly higher than the colocalization coefficient of 0.103 in the cell nucleus (blue), confirming the high driving and targeting ability of the probe Ir-visc to mitochondria.

[0069] Figure 3 b is the single-photon (OP) and two-photon (TP) confocal images of the iridium complex Ir-visc of the present invention after incubation with HepG2 cells, as shown in FIG. Figure 3 As shown in b, the OP (450 nm, 0.2 W / cm2) of iridium complex Ir-visc (10 μM) in HepG2 cells 2 ) and TP (840nm, 0.2W / cm 2Confocal imaging. Both OP and TP signals are emitted from the same location in the cell, indicating that Ir-visc is localized in mitochondria at different excitation wavelengths. Combined with the quantitative profile analysis of the cellular components, the TP emission signal is proportional to the square of the excitation power compared with OP imaging, so TP is excited more strongly in the limited space of the focal plane, thus showing better signal-to-noise ratio in imaging, and is more superior to biological imaging and diagnosis of deep tissues.

[0070] Figure 4 a is the confocal image of the iridium complex Ir-visc of the application for monitoring the change of mitochondrial phosphorescence in HepG2 cells before and after CCCP treatment, as shown in Figure 4 a, it is verified that the targeting of iridium complex Ir-visc to mitochondria is independent of MMP. Before confocal imaging, the mitochondria of HepG2 cells were treated with carbonyl cyanophenylhydrazone (CCCP) to damage the mitochondria and reduce the mitochondrial membrane potential, and then the treated HepG2 cells were incubated with Ir-visc (10 μM), and the results showed that Ir-visc accumulated in mitochondria and stably produced yellow phosphorescence signals, regardless of whether MMP was normal or damaged, and overlapped well with the commercial dye Mito-Lite Green.

[0071] Figure 4 b is the interaction diagram between the iridium complex Ir-visc of the application and the adjacent residues of the protein (OPA1), as shown in Figure 4 b, it is verified that the complex Ir-visc exists weak interactions such as π-anion interaction, π-alkyl interaction and hydrogen bond between the nonpolar amino acid residues Met60, Pro53, Ala95, the polar amino acid residues Ser247, Asn245, and the ionic amino acid residues Arg54, Arg50, Arg98, Glu102 of the mitochondrial protein OPA1 through molecular docking simulation. Therefore, Ir-visc can be used for long-term and high-precision mitochondrial visualization in the case of MMP deficiency by binding to the inner membrane protein of mitochondria and stably riveting in mitochondria independent of MMP.

[0072] Figure 5 a is the confocal image of the iridium complex Ir-visc of the application after incubation with tumor cells (HeLa cells and HepG2 cells) and normal cells (HUVEC cells and HL-7702 cells), respectively, as shown in Figure 5a shows that considering the viscosity activation characteristics of iridium complex Ir-visc, whether it can effectively distinguish cancer cells and normal cells. After co-incubation of iridium complex Ir-visc (10 μM) with normal cells (HUVEC cells and HL-7702 cells) and cancer cells (HeLa cells and HepG2 cells) for 10 min, confocal microscope imaging was performed. Iridium complex Ir-visc showed strong phosphorescence signal in both cancer cells, while the phosphorescence signal in both normal cells was very weak, which may be related to the higher viscosity of cancer cells than normal cells.

[0073] Figure 5 b is the relative phosphorescence intensity quantification image of iridium complex Ir-visc of the application after incubation with tumor cells (HeLa cells and HepG2 cells) and normal cells (HUVEC cells and 7702 cells), as shown in Figure 5 b shows that in order to quantify the ability of iridium complex Ir-visc to distinguish cancer cells and normal cells, the phosphorescence intensity ratio between cancer cells and normal cells is calculated. The phosphorescence intensity ratio of cancer cells to normal cells is about 2.90, indicating that Ir-visc can distinguish cancer cells from normal cells according to the viscosity difference between cancer cells and normal cells.

[0074] Figure 5 c is the confocal image of iridium complex Ir-visc of the application after treatment of HepG2 cells with Nystatin and HBSS, as shown in Figure 5 c shows that HepG2 cells were treated with Nystatin (10 μM) and serum-free HBSS (12 h), respectively, and then co-incubated with iridium complex Ir-visc (10 μM). Nystatin can cause mitochondrial swelling, thereby increasing mitochondrial viscosity; HepG2 cells treated with serum-free HBSS can cause cells to be in a state of starvation, and the viscosity of mitochondria increases. HepG2 cells treated with Nystatin and HBSS show strong yellow signal compared with the control group, indicating that iridium complex Ir-visc can well detect the viscosity changes induced by endogenous in living cells.

[0075] Figure 5 d is the relative phosphorescence intensity quantification image of iridium complex Ir-visc of the application after treatment of HepG2 cells with Nystatin and HBSS, as shown in Figure 5 d shows that by quantifying the phosphorescence intensity of iridium complex Ir-visc in Figure 5 c, the changes in viscosity in HepG2 cells can be more clearly reflected. It shows that Ir-visc can be used as a powerful tool for studying mitochondrial function and disease mechanism by dynamically analyzing the changes in mitochondrial viscosity.

[0076] Figure 6 a is the OP and TP confocal images of the iridium complex Ir-visc of the present invention on the tumor tissue and normal tissue areas of LLC tumor-bearing mice, as shown in Figure 6 As shown in Figure a, the ability of the iridium complex Ir-visc to selectively image tumor regions in living mice was evaluated. After establishing an LLC tumor-bearing mouse model, the iridium complex Ir-visc was dispersed in PBS (500 μM) and 50 μL was injected intratumorally into two different sites (tumor site and normal site) of the tumor-bearing mice. In the absence of Ir-visc injection, the mouse surface exhibited very low fluorescence background signal within the imaging window, indicating negligible interference from biological autofluorescence. After Ir-visc injection, weak phosphorescence signals were observed in normal tissue, while more pronounced phosphorescence signals were observed in tumor tissue under 450 nm and 808 nm excitation light sources. The OP and TP phosphorescence intensity ratios of cancer / normal tissue were 2.80 and 3.83, respectively, indicating that Ir-visc can effectively distinguish between tumor and normal tissue. Furthermore, this suggests that the strategy combining two-photon properties with sticky activation can significantly improve the accuracy of precise imaging of tumor tissue.

[0077] Figure 6 b is a diagram of the iridium complex Ir-visc of the present invention identifying tumor tissue and normal tissue by OP and TP imaging means, as shown in FIG. Figure 6 As shown in Figure b, the application of the iridium complex Ir-visc for tumor margin identification was investigated. To facilitate experimental observation, tissue with clear tumor boundaries was selected as the study specimen. After incubation of the tissue with Ir-visc (50 μM) for 2 hours, OP and TP imaging was performed. The captured OP and TP phosphorescence signals overlapped well with the cancerous tissue and were clearly demarcated from normal tissue, clearly demonstrating the difference between tumor and normal tissue.

[0078] Figure 6 c is the 3D phosphorescence depth image of OP and TP after the tumor tissue is treated with the iridium complex Ir-visc of the present invention, as shown in FIG. Figure 6 As shown in Figure c, after Ir-visc (50 μM) was co-incubated with tumor tissue from an LLC tumor-bearing mouse model for 2 hours, the tissue penetration depth of Ir-visc was assessed by three-dimensional phosphorescence imaging. Notably, the maximum depth of TP imaging reached 75 μm, significantly deeper than that of the OP group (35 μm). The specific recognition of tumors by Ir-visc and its ability to induce deep tissue imaging through near-infrared light make it a potential material for cancer diagnosis.

[0079] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An iridium complex, characterized in that The structural formula of the iridium complex is shown below:

2. The method for preparing an iridium complex according to claim 1, wherein The steps include: 3,4-diethoxybenzaldehyde and o-aminothiophenol were dissolved in ethanol, heated under reflux for reaction, and the product was filtered under reduced pressure and dried to obtain C ^ N ligand L1; In a nitrogen atmosphere and in a dark environment, the above C ^ N ligand L1 and iridium trichloride trihydrate, then add ethylene glycol monoethyl ether and water as solvent, heat reflux reaction, cool to room temperature, and filter the precipitated solid under reduced pressure to obtain the intermediate iridium chloride bridge M; Under nitrogen protection, a tetrahydrofuran solution of sodium hydride was added to a three-necked flask, and the mixture was slowly cooled to room temperature in an ice bath. A tetrahydrofuran solution of pyrazole was slowly added to form a light yellow solution, and then thionyl chloride was slowly added. The mixture was stirred at room temperature for reaction, and then 2-thiophenealdehyde and anhydrous cobalt chloride were added and heated under reflux for reaction. After cooling to room temperature, water was added, and the mixture was extracted with dichloromethane, dried over anhydrous magnesium sulfate overnight, and then dried by rotary evaporation. The N ^ N ligand L2; Under nitrogen and light-proof conditions, the intermediate iridium chloride bridge M, N ^ The N ligand L2 and ammonium hexafluorophosphate, a mixed solution of methanol and dichloromethane are used as solvents, heated under reflux for reaction, the reaction is stopped, cooled to precipitate a solid, and filtered under reduced pressure to obtain the target product iridium complex.

3. The method for preparing an iridium complex according to claim 2, wherein The molar ratio of the 3,4-diethoxybenzaldehyde to the 2-aminothiophenol is 1:1-1.

2.

4. The method for preparing an iridium complex according to claim 2, wherein The C ^ The molar ratio of the N ligand L1 to iridium trichloride trihydrate is 2-2.5:

1.

5. The method for preparing an iridium complex according to claim 2, wherein The volume ratio of ethylene glycol monoethyl ether to water in the mixed solvent is 3:

1.

6. The method for preparing an iridium complex according to claim 2, wherein The molar ratio of pyrazole, thionyl chloride and 2-thiophene aldehyde is 2:1:0.2-0.5; the molar ratio of sodium hydride:pyrazole is 1:1; and the molar ratio of anhydrous cobalt chloride:2-thiophene aldehyde is 1:3-3.

5.

7. The method for preparing an iridium complex according to claim 2, wherein The intermediate iridium chloride bridge M, N ^ The molar ratio of the N ligand L2 to ammonium hexafluorophosphate is 1:2-3:2-4.

8. The method for preparing an iridium complex according to claim 2, wherein The volume ratio of methanol to dichloromethane in the mixed solvent is 1:

1.

9. The method for preparing an iridium complex according to any one of claims 2 to 8, characterized in that: In steps S1-S4, the conditions for heating reflux reaction are: C ^ In the preparation steps of N ligand L1: the temperature is 70-80°C and the time is 6-8h; In the preparation steps of the intermediate iridium chloride bridge M: the temperature is 110-120°C and the time is 24-30 hours; N ^ In the preparation steps of N ligand L2: the temperature is 70-80°C and the time is 10-12h; In the preparation steps of the iridium complex, the temperature is 75 to 80° C. and the time is 12 to 16 hours.

10. Use of the iridium complex according to claim 1, characterized in that: The iridium complex is used to prepare a composition, a probe or a kit for dynamic detection of mitochondrial viscosity and / or tumor differentiation.

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