Iridium complex for dynamic mitochondrial viscosity detection and tumor distinguishing as well as preparation method and application of iridium complex

Abstract

The invention relates to the technical field of biological medicine, and discloses an iridium complex for dynamic detection of mitochondrial viscosity and tumor distinguishing and a preparation method and application thereof, the structural formula of the iridium complex is as follows: # imgabs0. The iridium complex Ir-visc is based on an electrostatic adsorption effect and a double-locking targeting strategy of probe-protein docking, does not depend on MMP, and has a good application prospect. The mitochondria in the anchoring cell is stabilized; the compound shows excellent high-viscosity activated single-photon (OP) and two-photon (TP) phosphorescence characteristics, is not interfered by other factors in a living body, and can be used for monitoring the change of viscosity in a complex biological system; the fluorescent probe can be used as a mitochondrial phosphorescent probe for distinguishing tumor cells from normal cells and monitoring changes of cellular mitochondrial viscosity under the stimulation of hunger and antifungal drug nystatin; the method not only has strong tumor tissue recognition capability, but also has deep tissue penetration and living body imaging capabilities.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and discloses an iridium complex material for dynamic detection of mitochondrial viscosity and tumor differentiation and a preparation method thereof, specifically an iridium complex with high quantum yield under viscosity activation, which can be used to selectively illuminate cancer cells and monitor mitochondrial viscosity, and a preparation method and use thereof. Background Art

[0002] Fluorescence imaging technology has the advantages of being non-invasive, ultra-sensitive, highly selective, and easy to use, and has shown great potential and application prospects 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 wastes and promoting signal transduction and other biological processes. Abnormal changes in viscosity are closely related to malignant tumors, which are characterized by the uncontrolled growth of cancer cells and the accumulation of high-viscosity metabolites, especially in mitochondria, where mitochondrial viscosity is significantly increased compared with normal cells. Therefore, mitochondrial viscosity may be a potential target for designing activatable probes to identify cancer cells. Using mitochondrial viscosity as a link, sensing the microenvironment around early tumor tissue is crucial for tracking tumorigenesis and detecting tumors. However, the high dependence of most current viscosity-activated mitochondrial probes on mitochondrial membrane potential (MMP) as well as the short luminescence lifetime and poor tissue penetration depth seriously hinder their widespread application. Therefore, it is crucial to develop MMP-independent mitochondrial viscosity-activated probes to accurately and reliably locate and quantitatively monitor mitochondrial viscosity in the fields of biological research, cancer diagnosis, and drug development.

[0003] Octahedral luminescent cyclometalated iridium (III) complexes [Ir(C^N)2(N^N)]+ are widely used in bioimaging and biosensing due to their high quantum yield, large Stokes shift, long luminescence lifetime, and good photostability. In addition, iridium complexes can be easily modified with ligands to obtain excellent two-photon absorption (TPA) properties, thus having the characteristics of deep tissue penetration, low photobleaching, phototoxicity, and photodamage, and weak autofluorescence. Summary of the invention

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

[0005] The design concept of the present invention includes: the present invention utilizes the double-locked mitochondrial targeting strategy to rationally design an iridium complex (Ir-visc) with viscosity-activated TPA absorption activity to meet the above challenges. First, a benzothiazole derivative is used as a C^N ligand, and the structure of the benzothiazole platform is modified to regulate the biological activity and enhance the binding ability of the probe protein molecule; secondly, the electron-rich thiophene ring is used as a molecular rotor, and is connected to the pyrazole ring through a single bond to form an N^N ligand, thereby regulating the charge distribution and viscosity activation 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 a negative membrane potential. In addition, the synergistic effect of the polyaromatic ring and the positive charge enables Ir-visc to bind to mitochondrial membrane proteins by forming non-covalent bonds (such as hydrogen bonds, ionic bonds, and van der Waals forces).

[0006] In a first aspect, the present invention provides an iridium complex, the structural formula of the complex is as follows:

[0007]

[0008] In a second aspect, the present invention also provides a method for preparing an iridium complex, comprising the following steps:

[0009] C ^ Synthesis of N ligand L1: 3,4-diethoxybenzaldehyde and o-aminothiophenol were dissolved in ethanol, heated under reflux to react, and a white filamentous product was obtained. The product was filtered under reduced pressure and dried to obtain C ^ N ligand L1;

[0010] Synthesis of intermediate iridium chloride bridge M: Add the above C ^ N ligand and iridium trichloride trihydrate, then add ethylene glycol monoethyl ether and water as solvent, heat to reflux for reaction, cool to room temperature, precipitate orange solid, and filter under reduced pressure to obtain intermediate iridium chloride bridge M;

[0011] N ^ Synthesis of N ligand L2: Under nitrogen protection, add a tetrahydrofuran solution of sodium hydride to a three-necked flask, slowly cool to room temperature in an ice bath, slowly add a tetrahydrofuran solution of pyrazole to form a light yellow solution, then slowly add dichlorothionyl to obtain a yellow-white suspension, stir at room temperature to react, then add 2-thiophenealdehyde and anhydrous cobalt chloride to heat under reflux to react, cool to room temperature, add water, then extract with dichloromethane, dry over night with anhydrous magnesium sulfate, spin dry, and purify by column chromatography to obtain N ^ N ligand L2;

[0012] Synthesis of complex Ir-visc: In nitrogen and light-proof conditions, add intermediate iridium chloride bridge M, N ^The N ligand L2 and ammonium hexafluorophosphate, a mixed solution of methanol and dichloromethane are used as solvents, and after heating and reflux reaction, the reaction is stopped and cooled to precipitate a yellow solid, which is filtered under reduced pressure to obtain the target product iridium complex.

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

[0014] Furthermore, C ^ The molar ratio of the 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] Furthermore, 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.

[0016] Further, the intermediate iridium chloride bridge M, N ^ The molar ratio of the 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 for the heating reflux reaction are:

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

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

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

[0021] In the preparation steps of the iridium complex: the temperature is 75-80° C. and the time is 12-16 hours.

[0022] In a third aspect, the present invention also provides a use of an iridium complex, wherein the iridium complex is used to prepare a composition, a probe or a kit for dynamic detection of mitochondrial viscosity and / or tumor differentiation.

[0023] Technical effects and advantages of the present invention:

[0024] 1. The iridium complex Ir-visc is based on a dual-locking targeting strategy of electrostatic adsorption and probe-protein docking. It is independent of MMP and can stably anchor mitochondria in cells.

[0025] 2. The iridium complex Ir-visc exhibits excellent high-viscosity activated single-photon (OP) and two-photon (TP) phosphorescence properties, and is not interfered by other factors in the organism, and 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 of 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 partly become obvious from the description, or be understood by implementing 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 briefly introduces 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 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 nuclear magnetic resonance 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 a 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 phosphorescence intensity (LogI) of the iridium complex Ir-visc of the present invention at different viscosities (Logη) and 575nm575nm )’s linear correlation graph;

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

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

[0038] 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) diagram;

[0039] 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 cell nucleus dye (Hochest 33342);

[0040] 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 co-incubation with HepG2 cells;

[0041] Figure 4 a is a confocal image of the iridium complex Ir-visc of the present invention used to monitor the changes in mitochondrial phosphorescence in HepG2 cells before and after CCCP treatment;

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

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

[0044] Figure 5 b is a quantitative image of the relative phosphorescence intensity after the iridium complex Ir-visc of the present invention was incubated with tumor cells (Hela cells and HepG2 cells) and normal cells (HUVEC cells and HL-7702 cells);

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

[0046] Figure 5 d is a quantitative image of the relative phosphorescence intensity of HepG2 cells after the iridium complex Ir-visc of the present invention is treated with Nystatin and HBSS respectively;

[0047] 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 area of ​​LLC tumor-bearing mice;

[0048] Figure 6 b is a diagram showing the iridium complex Ir-visc of the present invention distinguishing tumor tissue and normal tissue by OP and TP imaging methods;

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

[0050] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] The flowcharts shown in the accompanying drawings are only exemplary and do not necessarily include all the steps. For example, some steps may be decomposed, while some steps may be combined or partially combined, so the actual execution order may change according to the actual situation.

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

[0053] Example 1: Preparation of Iridium Complex

[0054] Step S1, synthesis of C^N ligand L1: 1.94 g (10.0 mmol) of 3,4-diethoxybenzaldehyde and 1.25 g (10.0 mmol) of 2-aminothiophenol were added to a 100 mL round-bottom flask, dissolved with an appropriate amount of ethanol (just dissolve), refluxed at 75°C for 6 h, cooled and precipitated a white needle-like product, and dried to obtain 2.1 g of C^N ligand L1, with a yield of 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 intermediate iridium chloride bridge M: under nitrogen and light protection, add 0.65 g (2.2 mmol) of L1 and 0.35 g (1.0 mmol) of iridium (III) chloride trihydrate into a 50 mL Shrek bottle, add 30 mL of ethylene glycol monoethyl ether and 5 mL of water as a mixed solvent, heat to 110 ° C and reflux for 24 h, cool to precipitate an orange solid, and obtain 0.61 g of intermediate iridium chloride bridge M with a yield of 74%.

[0057]

[0058] Step S3, synthesis of N^N ligand L2: Under nitrogen protection, 1.16g (48mmol) NaH was added to a three-necked flask containing 50mL THF (refined), and the mixture was slowly cooled to 0°C in an ice bath. 15mL of a THF solution of 3.27g (48mol) pyrazole was slowly added within 15min, and stirred for 30min to form a light yellow solution. Then 2.91g (24mmol) SOCl2 was slowly added to obtain a yellow-white suspension, and the ice bath was removed and reacted at room temperature for 40min. Then 0.54g (4.8mmol) 2-thiophene aldehyde and 0.19g (1.4mmol) anhydrous cobalt chloride were added to the above system, refluxed for 10h, cooled to room temperature, 40mL water was added, stirred for 10min, extracted with dichloromethane, dried over anhydrous magnesium sulfate overnight, spin-dried, and purified by column chromatography with petroleum ether: ethyl acetate (5:1) to obtain 0.58g 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), with a yield of 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 spectral 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, such as Figure 2As shown in 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 iridium complexes in different polarity solution states have low phosphorescence emission. In glycerol, the high viscosity environment restricts the free rotation of Ir-visc, and an obvious emission peak enhancement is observed at 575nm.

[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 in the ratio of glycerol to methanol 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%), 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 of the present invention at different viscosities (Logη) and 575nm 575nm ) is a 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 prove that the generation of Ir-visc emission signal is only related to the change of 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, such as Figure 2 As shown in (d), the phosphorescence emission spectrum of Ir-visc is 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 f shows the TP absorption cross sections of the iridium complex Ir-visc in methanol and glycerol under the conditions of excitation wavelength of 680-1000nm, pulse duration of 140fs and power of 500mW. Under 840nm light excitation, Ir-visc has the best TPA cross section, and the TPA cross section in glycerol (76.33GM) is nearly 4.2 times higher than that in methanol (18.23GM), indicating that it has the potential to become 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 =430-470nm) 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, mitochondrial dye (Mito-Lite Green) and cell nucleus dye (Hochest 33342), as shown in FIG. 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 co-incubation with HepG2 cells, as shown in Figure 3 b shows the OP (450 nm, 0.2 W / cm2) of the iridium complex Ir-visc (10 μM) in HepG2 cells. 2 ) and TP (840nm, 0.2W / cm 2) confocal imaging. Both OP and TP signals are emitted from the same position in the cell, indicating that Ir-visc is localized to mitochondria at different excitation wavelengths. Combined with quantitative atlas analysis of cell components, compared with OP imaging, TP emission signals are proportional to the quadratic curve of excitation power, so TP is more strongly excited in the limited space of the focal plane, thus showing a better signal-to-noise ratio in imaging, which is superior to biological imaging and diagnosis of deep tissues.

[0070] Figure 4 a is a confocal image of the iridium complex Ir-visc of the present invention used to monitor the changes in mitochondrial phosphorescence in HepG2 cells before and after CCCP treatment, such as Figure 4 As shown in a, it was verified that the targeting of the iridium complex Ir-visc to mitochondria was independent of MMP. Before confocal imaging, the mitochondria of HepG2 cells were treated with carbonyl cyanide-p-chlorophenylhydrazone (CCCP) to damage the mitochondria and reduce the mitochondrial membrane potential. The treated HepG2 cells were then co-incubated with Ir-visc (10 μM). The results showed that regardless of whether MMP was normal or damaged, Ir-visc accumulated in the mitochondria and stably produced yellow phosphorescent signals, which overlapped well with the commercially stained Mito-Lite Green.

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

[0072] Figure 5 a is a confocal image of the iridium complex Ir-visc of the present invention after incubation with tumor cells (HeLa cells and HepG2 cells) and normal cells (HUVEC cells and HL-7702 cells), as shown in FIG. Figure 5As shown in a, considering the viscosity activation properties of the iridium complex Ir-visc, it was investigated whether it can effectively distinguish cancer cells from normal cells. Iridium complex Ir-visc (10 μM) was incubated with normal cells (HUVEC cells and HL-7702 cells) and cancer cells (HeLa cells and HepG2 cells) for 10 minutes, and then confocal microscopy imaging was performed. Iridium complex Ir-visc showed strong phosphorescence signals in both cancer cells, while the phosphorescence signals in both normal cells were extremely weak, which may be related to the higher viscosity of cancer cells than normal cells.

[0073] Figure 5 b is the quantitative image of the relative phosphorescence intensity after the iridium complex Ir-visc of the present invention was incubated with tumor cells (HeLa cells and HepG2 cells) and normal cells (HUVEC cells and 7702 cells), as shown in FIG. Figure 5 As shown in b, in order to quantify the ability of the iridium complex Ir-visc to distinguish cancer cells from normal cells, the phosphorescence intensity ratio between cancer cells and normal cells was calculated. Phosphorescence quantitative analysis showed that the fluorescence intensity ratio between cancer cells and normal cells was about 2.90, indicating that Ir-visc can distinguish cancer cells from normal cells by generating phosphorescence differences based on the viscosity differences between cancer cells and normal cells.

[0074] Figure 5 c is a confocal image of HepG2 cells treated with the iridium complex Ir-visc of the present invention, nystatin (Nystatin) and serum-free medium (HBSS), respectively. Figure 5 As shown in c, HepG2 cells were treated with nystatin (10 μM) and serum-free high glucose medium (HBSS, 12 h), respectively, and then incubated with the 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 starved state, and mitochondrial viscosity increases. HepG2 cells treated with Nystatin and HBSS showed a strong yellow signal compared with the control group, indicating that the iridium complex Ir-visc can well detect changes in viscosity induced endogenously in living cells.

[0075] Figure 5 d is a quantitative image of the relative phosphorescence intensity of HepG2 cells after the iridium complex Ir-visc of the present invention is treated with Nystatin and HBSS respectively. Figure 5 d, through Figure 5 The phosphorescence intensity of the iridium complex Ir-visc in c is quantified to more clearly reflect the changes in viscosity in HepG2 cells. This shows that Ir-visc can be used as a powerful tool to study mitochondrial function and disease mechanisms through dynamic analysis of 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 area of ​​LLC tumor-bearing mice, as shown in Figure 6 As shown in a, the ability of the iridium complex Ir-visc to selectively image tumor areas in living mice was evaluated. After preparing the LLC tumor-bearing mouse model, the iridium complex Ir-visc was dispersed in PBS (500 μM), and 50 μL was injected into two different parts of the tumor-bearing mice (tumor site and normal site) by intratumoral injection. In the absence of Ir-visc injection, the mouse surface showed a very low fluorescence background signal within the imaging window, indicating that the interference caused by biological autofluorescence was negligible. After Ir-visc injection, under 450nm and 808nm excitation light sources, it was observed that the phosphorescence signal of normal tissue was weak, while the phosphorescence signal of tumor tissue was obvious. The OP and TP phosphorescence intensity ratios of cancer / normal tissue were 2.80 and 3.83, indicating that Ir-visc can effectively distinguish tumors from normal tissues. At the same time, it is suggested that the strategy combining two-photon properties and sticky activation can significantly improve the accuracy of precise imaging of tumor tissues.

[0077] Figure 6 b is a diagram of the iridium complex Ir-visc of the present invention distinguishing tumor tissue and normal tissue by OP and TP imaging means, such as Figure 6 As shown in Figure b, the application of iridium complex Ir-visc in tumor edge recognition was studied. To facilitate experimental observation, tissues with clear tumor boundaries were selected as research specimens. After Ir-visc (50 μM) was co-incubated with the tissue for 2 hours, OP and TP imaging was performed. The collected OP and TP phosphorescence signals overlapped well with the cancer tissue and were clearly demarcated from the normal tissue, clearly showing the difference between tumor and normal tissue.

[0078] Figure 6 c is a three-dimensional phosphorescence depth image of OP and TP after the tumor tissue is treated with the iridium complex Ir-visc of the present invention. Figure 6 As shown in c, after Ir-visc (50 μM) was co-incubated with tumor tissue of LLC tumor-bearing mouse model for 2 h, the tissue penetration depth of Ir-visc was evaluated by three-dimensional phosphorescence imaging. It is worth noting that the maximum depth of TP imaging reached 75 μm, which was significantly deeper than that of OP group (35 μm). The specific recognition of Ir-visc for tumors and the ability of near-infrared light-induced deep tissue imaging 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, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to 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 protection scope 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, add the above C ^ N ligand L1 and iridium trichloride trihydrate, then add ethylene glycol monoethyl ether and water as solvent, heat to reflux for reaction, cool to room temperature, and filter the precipitated solid under reduced pressure to obtain the intermediate iridium chloride bridge M; Under nitrogen protection, add a tetrahydrofuran solution of sodium hydride to a three-necked flask, slowly cool to room temperature in an ice bath, slowly add a tetrahydrofuran solution of pyrazole to form a light yellow solution, slowly add thionyl chloride, stir at room temperature for reaction, then add 2-thiophenealdehyde and anhydrous cobalt chloride, heat under reflux for reaction, cool to room temperature, add water, extract with dichloromethane, dry over anhydrous magnesium sulfate overnight, spin dry, and purify to obtain N ^ N ligand L2; Under nitrogen and light-proof conditions, add 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 solids, and filtered under reduced pressure to obtain the target product iridium complex.

3. The method for preparing an iridium complex according to claim 2, characterized in that: 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, characterized in that: The C ^ The molar ratio of the N ligand L1 to iridium trichloride trihydrate is 2 to 2.5:

1.

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

8. The method for preparing an iridium complex according to claim 2, characterized in that: 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 24-30h; 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 75-80° C. and the time is 12-16 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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