CN110857311B - Ring metal iridium complex with tumor hypoxia sensing and optical activity and application thereof - Google Patents
Ring metal iridium complex with tumor hypoxia sensing and optical activity and application thereof Download PDFInfo
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- FHNFHKCVQCLJFQ-UHFFFAOYSA-N xenon atom Chemical compound [Xe] FHNFHKCVQCLJFQ-UHFFFAOYSA-N 0.000 description 1
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- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
- C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
- C07F15/0033—Iridium compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
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Abstract
The invention designs a novel cyclometalated iridium complex based on Ir (III), which has the following structure:
or
Description
Technical Field
The invention belongs to the field of metal complexes, and particularly relates to a cyclometalated iridium complex with tumor hypoxia sensing and optical activity and application thereof.
Background
The concentration of oxygen in normal tissue cells is about 5-20%, while in solid tumors the concentration of oxygen is below 4%, even reaching 0% in regions distant from the tumor vessels. Hypoxic tumor tissue is resistant to conventional radiation therapy. Similar to radiotherapy, the action mechanism of photodynamic therapy also depends on the generation of singlet oxygen to kill tumor cells, and most of the photosensitizers reported at present are oxygen-dependent. Under hypoxic conditions, the PDT effect of the classical photosensitizer, hematoporphyrin derivative, is obviously inhibited, the critical value of the oxygen concentration of PDT activity is about 15-35mm Hg, and the photodynamic efficiency of the photosensitizer Foscan is completely inhibited. For hypericin and phthalocyanine photosensitizers, their toxicity to cells through photodynamic effects is also dependent on oxygen concentration. In addition, the PDT treatment process consumes oxygen in the process of generating singlet oxygen, so that oxygen deficiency is caused, and the treatment effect is influenced.
The development of photosensitizers with type I photosensitization mechanisms is one solution to the decrease in photodynamic efficiency caused by tumor hypoxia. The cyclometalated iridium (III) complexes also play an important role as an important class thereof. As a photosensitizer for photodynamic therapy, iridium (III) complexes can achieve high Reactive Oxygen Species (ROS) yields through modulation of their excited states. In addition, as photosensitizers for photodynamic therapy, the generation of active oxygen species of cyclometalated iridium (III) complexes can be via either electron progression (type I) or energy transfer (type II). The McFarland group reported a photosensitizer TLD1433 based on a ruthenium (II) polypyridyl complex of transition metals for photodynamic antimicrobial therapy (PACT), which does not emit light in all solvents (quantum yield < 0.05%), indicating the possible presence of a very important excited state energy relaxation pattern in type I photosensitizers. TLD1433 had better photodynamic antibacterial activity at normoxic conditions (an order of magnitude reduction log10reductions of 6.7 for Staphylococcus aureus). On the other hand, in the case of hypoxia, the photosensitizer can produce photodynamic antibacterial effect (6.8 log10reductions on staphylococcus aureus) based on type I phototherapy mechanism. In addition, TLD1433 is found to have certain potential in the aspect of photodynamic anti-tumor in subsequent research, and at present, the photosensitizer enters the clinical stage I research stage and has a good effect in the aspect of photodynamic therapy of bladder cancer.
On the other hand, based on the principle that the fluorescence intensity difference is formed by the difference of the microenvironments of part of photosensitizers in normal tissues and cancer tissues, pathological tissues can be effectively distinguished, and the Photodynamic diagnosis (PDD) is based on the principle. Because oxygen has a quenching effect on phosphorescence, the oxygen partial pressure in a hypoxic region inside the tumor is very low, and phosphorescence cannot be quenched, so that the tumor hypoxia detection and imaging can be realized.
Disclosure of Invention
The invention designs a novel photosensitizer based on Ir (III), which can realize direct and reversible detection of oxygen concentration by utilizing the oxygen-dependent phosphorescence quenching characteristic. Meanwhile, the photosensitizer shows high antitumor activity in both normoxic and hypoxic modes of photodynamic therapy of tumors.
The specific technical scheme of the invention is as follows:
a cyclometalated iridium complex is characterized by having the following structure:
the above cyclometalated iridium complex may further be combined with an anion selected from the group consisting of Cl-、Br-，I-，NO3 -Or PF6 -。
Specific cyclometalated iridium complexes of the invention are selected from
The invention also aims to disclose the application of the cyclometalated iridium complex in preparing a photosensitizer.
Preferably, the cyclometalated iridium complex is a photosensitizer associated with photodynamic therapy. The cyclometalated iridium complex can be used as a photosensitizer to be applied to cell confocal imaging.
One preferable application in the applications is the application of the cyclometalated iridium complex in preparing antitumor drugs.
The tumor is selected from melanoma, breast cancer, cervical cancer, gastric cancer, lung cancer or liver cancer.
One preferable application of the above applications is an application of the cyclometalated iridium complex in preparation of a living cell oxygen detection reagent. The cyclometalated iridium complex carries out reversible phosphorescence detection and identification on oxygen concentration.
The cyclometalated iridium complex can be prepared by the following method:
the iridium complex is chloro-bridged dimer Ir2(iqbt)4Cl2(commercially available), compound L3 or compound L4 (obtainable by the methods disclosed in references j.liu, y.chen, g.li, p.zhang, c.jin, l.zeng, l.ji, h.chao, Biomaterials,2015,56, 140) and dichloromethaneAnd (3) placing the mixed solvent of the alkane and the methanol into a reaction container, and heating and refluxing under the protection of argon. After the reaction was completed, the reaction mixture was cooled to room temperature, and ten-fold equivalent of ammonium hexafluorophosphate was added. The suspension was stirred and filtered to remove insoluble inorganic salts. The solvent was evaporated under reduced pressure and column chromatography was carried out to give Mito-IrL3 or IrL 4.
The invention has the advantages that:
the cyclometalated iridium (III) complex has activity on tumor hypoxia sensing and photodynamic therapy, and is suitable for hypoxia detection and reversible phosphorescence detection and identification on oxygen concentration under physiological conditions. Has good stability in a physiological pH range and can be completely used for the contrast of a biological system. Can be used for imaging in cells or organisms for a long time without photobleaching. Has better mitochondrion targeting performance. Has obvious hypoxia selectivity on cell imaging. Is suitable for photodynamic therapy under the condition of hypoxia. The cyclometalated iridium (III) complex can affect mitochondria under the irradiation of light under normal oxygen to cause the reduction of membrane potential; and does not cause the reduction of mitochondrial membrane potential under dark conditions, and can be used as photosensitizer for photodynamic therapy.
According to the invention, the cyclometalated iridium complex with high photodynamic therapy activity under both normal oxygen and hypoxic conditions is constructed, the problem of reduced photodynamic therapy activity under the hypoxic condition of a conventional photosensitizer is solved, and researches show that the photodynamic therapy activity of the complex is possibly related to a type I photosensitization mechanism. Meanwhile, mitochondrial targeting of the iridium complex is realized by introducing a mitochondrial targeting group into the complex, and the mitochondrial targeting capability further improves the photodynamic therapy activity of the complex, which shows that the subcellular organelle targeting photosensitizer design strategy can effectively improve the photodynamic therapy capability of the photosensitizer. In addition, the complex can sensitively and reversibly respond to the change of oxygen concentration, has potential application to future tumor diagnosis and is expected to realize accurate site-specific treatment on tumors.
Drawings
FIG. 1 shows the phosphorescence emission spectra of the complexes Mito-IrL3 and IrL4 under different oxygen concentrations and the decay curves of the phosphorescence emission peaks thereof with the increase of the oxygen concentration.
FIG. 2 Stern-Volmer curves quenched by oxygen-pair complexes Mito-IrL3 and IrL4
FIG. 3. phosphorescence intensity at 685nm for complexes Mito-IrL3 and IrL4 was varied at both 0% and 21% oxygen concentrations.
FIG. 4 shows the variation of the intensity of the maximum emission peak at 685nm under different pH conditions of the complexes Mito-IrL3 and IrL 4.
FIG. 5 shows the change of phosphorescence intensity of the complexes Mito-IrL3 and IrL4 at the maximum emission wavelength under different illumination conditions.
FIG. 6. light complexes Mito-IrL3 and IrL4 cause DPBF to exhibit absorption values at 413nm as a function of time.
FIG. 7. distribution of the complex Mito-IrL3 in mitochondria and the intensity distribution of Mito-IrL3 and Mito-Tracker Deep Red 633 in cells.
FIG. 8 is a graph of laser confocal imaging of the complexes Mito-IrL and IrL4 at different oxygen concentrations in MCF-7 cells.
FIG. 9 shows the survival rate of MCF-7 cells after the complexes Mito-IrL3 and IrL4 are incubated with MCF-7 cells under the conditions of normal oxygen and hypoxia and are irradiated by light, and the survival rate of the MCF-7 cells changes along with time.
Figure 10 is a confocal image of the fluorescence change of JC-1 after the complexes Mito-IrL3 and IrL4 are incubated with JC-1 under the conditions of normal oxygen and hypoxia.
Detailed Description
The invention is described in further detail below with reference to the accompanying drawings.
In order to better understand the contents of the present patent, the following further illustrates the technical solution of the present invention by specific examples. However, these examples do not limit the present invention.
EXAMPLE 1 Synthesis of Complex Mito-IrL3
Taking the synthesized iridium complex chlorine bridge dimer Ir2(iqbt)4Cl2(154mg, 0.1mmol), L3(157mg, 0.2mmol) and a mixed solvent of dichloromethane and methanol (12mL, 2:1, vol.) were placed in a reaction vessel and heated under reflux for 6h under an argon atmosphere. After the reaction was completed, the reaction mixture was cooled to room temperature, and ten-fold equivalent of ammonium hexafluorophosphate was added. The suspension was stirred for 15min and filtered to remove insoluble inorganic salts. The solvent was evaporated under reduced pressure and column chromatography afforded Mito-IrL3 as a dark red solid (56%).1H NMR(400MHz，CD3CN-d3)δ9.21(dd，J＝8.3，1.4Hz，1H)，9.07(dd，J＝13.3，8.8Hz，2H)，8.30(dd，J＝5.1，1.4Hz，1H)，8.15(dd，J＝5.1，1.2Hz，1H)，7.97–7.80(m，12H)，7.75–7.65(m，18H),7.55(d，J＝8.8Hz，2H)，7.45–7.34(m，3H)，7.24–7.13(m，4H)，6.84-6.81(m，2H)，6.70(q，J＝7.6，7.1Hz，2H)，6.22–6.17(m，2H)，4.01(t，J＝6.0Hz，2H)，3.29–3.15(m，2H)，1.85-1.77(m，2H)，1.24(s，2H).13C NMR(101MHz，CD3CN-d3)δ166.20，161.08，156.64，156.19，155.54，151.22，150.32，145.50，145.42，145.30，144.49，142.98，142.95，138.20，138.16，138.12，137.74，137.12，137.06，136.16，136.13，134.65，134.56，134.01，133.64，132.01，131.95，131.92，131.88，131.69，131.32，131.19，130.32，130.29，129.66，129.56，128.98，128.67，128.64，127.76，127.73，127.61，127.46，127.42，126.73，125.89，125.85，125.53，125.49，123.62，123.55，123.51，122.74，120.84，120.80，119.61，115.33，67.36，30.22，30.06，22.40，21.89，19.74，19.70。
Example 2 Synthesis of the Complex IrL4
Taking the synthesized iridium complex chlorine bridge dimer Ir2(iqbt)4Cl2(154mg, 0.1mmol), L4(89mg, 0.2mmol) and a mixed solvent of dichloromethane and methanol (12mL, 2:1, volume ratio) were placed in a reaction vessel, and under an argon atmosphere, addedAnd thermally refluxing for 6 h. After the reaction was completed, the reaction mixture was cooled to room temperature, and ten-fold equivalent of ammonium hexafluorophosphate was added. The suspension was stirred for 15min and filtered to remove insoluble inorganic salts. The solvent was evaporated under reduced pressure and column chromatography afforded IrL4 as a dark red solid (61%).1H NMR(400MHz，CD3CN-d3)δ9.13(d，J＝8.3Hz，1H)，9.04(dd，J＝14.7，8.5Hz，2H)，8.25(d，J＝4.4Hz，1H)，8.12(d，J＝4.4Hz，1H)，7.96–7.72(m，9H)，7.71–7.51(m，6H)，7.48(d，J＝8.7Hz，2H)，7.42–7.32(m，3H)，7.18-7.11(m，4H)，6.78(d，J＝8.7Hz，2H)，6.69-6.64(m，2H)，6.19(t，J＝7.1Hz，2H)，3.91(t，J＝6.5Hz，2H)，1.67(dt，J＝13.8，6.1Hz，2H)，1.49–1.35(m，2H)，0.92(t，J＝7.4Hz，3H).13C NMR(101MHz，CD3CN-d3)δ166.19，161.47，156.63，156.20，155.55，151.13，150.23，145.42，145.24，144.48，142.97，142.91，138.13，137.72，137.10，137.04，133.99，133.62，131.89，131.65，130.29，130.26，129.59，129.51，128.89，128.65，128.62，128.53，127.71，127.54，127.41，126.72，125.87，125.82，125.50，125.47，123.59，123.52，123.47，122.28，120.85，115.25，68.72，31.84，19.84，14.08.
Preparation of Complex Mito-IrL3 and IrL4 test solutions
Accurately weighing the complex Mito-IrL3 and IrL4, respectively transferring into a 5mL volumetric flask, diluting with DMSO to constant volume to obtain a solution with a concentration of 1.0 × 10-3Transferring 25 mu L of the solution into a 5mL volumetric flask, and preparing the solution with DMSO and PBS buffer solution with pH of 7.40 to constant volume to obtain the solution with the concentration of 1.0 multiplied by 10-5The ultraviolet spectrum of the solution of mol/L is measured. The fluorescence spectrum was measured using the maximum absorption wavelength of the ultraviolet spectrum as the excitation wavelength of the fluorescence spectrum.
EXAMPLE 3 study of the response of the complexes Mito-IrL3 and IrL4 to oxygen concentration
The phosphorescent response of 10. mu.M complexes Mito-IrL3 and IrL4 to oxygen concentration was studied in toluene.
The experimental results are shown in FIG. 1, in which FIG. 1a shows the phosphorescence emission spectrum of the complex Mito-IrL3 at different oxygen concentrations, and FIG. 1c shows the decay curve of the phosphorescence emission peak intensity at 685nm of the Mito-IrL3 toluene solution (10 μ M) with the increase of the oxygen concentration. FIG. 1b shows the phosphorescence emission spectrum of the complex IrL4 at different oxygen concentrations, and FIG. 1d shows the decay curve of the phosphorescence emission peak intensity at 685nm of the IrL4 toluene solution (10 μ M) with the increase of the oxygen concentration.
As can be seen from FIG. 1a, phosphorescence intensity response experiment of the oxygen concentration carried out by the complex Mito-IrL3 proves that the probe can respond to the fluctuation of the oxygen concentration. The near infrared phosphorescence emission peak intensity of the complex Mito-IrL3 at 685nm increases nonlinearly with decreasing oxygen concentration (FIG. 1 c). In the solution without oxygen and saturated with oxygen, the intensity ratio of the emission peak of the probe can reach 20.4. The decay curve of the emission peak intensity with the increase of oxygen concentration shows that the probe is particularly sensitive to oxygen partial pressure under low oxygen concentration, and under the low oxygen condition, a small change of the oxygen concentration can cause a sharp increase of the emission intensity of the probe, for example, the oxygen concentration is reduced from 2% to 0%, which can result in the increase of the probe intensity by about 40%, so that the probe is particularly suitable for hypoxia detection under physiological conditions due to the small oxygen concentration difference between tissues under physiological conditions.
As can be seen from FIG. 1b, the phosphorescence intensity response experiment of the oxygen concentration carried out by the complex IrL4 proves that the probe can also carry out similar response to the fluctuation of the oxygen concentration. As can be seen from the figure, the emission peak position of the probe does not change under the conditions of different oxygen concentrations. However, as the oxygen concentration increases, the emission intensity of IrL4 decreases significantly and non-linearly. The emission intensity of IrL4 was 3.5% in the case of pure oxygen gas and pure argon gas. Under hypoxic conditions, the probe is also very sensitive to oxygen partial pressure, and small changes in oxygen concentration can cause large changes in the emission intensity of the probe.
Example 4 oxygen sensing Stern-Volmer of the complexes Mito-IrL3 and IrL4
The oxygen concentration has dynamic quenching phenomenon to the phosphorescence intensity and luminescence lifetime of the complexes Mito-IrL3 and IrL 4. The present invention further investigated the Stern-Volmer curves quenched by oxygen for the complexes Mito-IrL3 and IrL 4.
The experimental results are shown in FIG. 2, in which FIG. 2a is a phosphorescence intensity quenching Stern-Volmer curve of the oxygen-complex Mito-IrL3, FIG. 2b is an emission lifetime quenching Stern-Volmer curve of the oxygen-complex Mito-IrL3, FIG. 2c is a phosphorescence intensity quenching Stern-Volmer curve of the oxygen-complex IrL4, and FIG. 2d is an emission lifetime quenching Stern-Volmer curve of the oxygen-complex IrL 4.
Both Mito-IrL3 and IrL4 exhibited very sensitive characteristics to changes in oxygen concentration. As can be seen from the graph, the dynamic quenching constant K of Mito-IrL3SVRespectively account for 0.17964%-1And 0.11309%-1Dynamic quenching constant K of IrL4SVRespectively account for 0.19307%-1And 0.18495%-1This high sensitivity is important for oxygen sensing materials given the small differences in oxygen content between tumor microenvironment and normal physiological microenvironment. Meanwhile, Stern-Volmer curves of Mito-IrL3 and IrL4 are shown to have good linear relationship.
Example 5 oxygen response reversibility study of the complexes Mito-IrL3 and IrL4
The hypoxia of the tumor is an important characteristic of a malignant solid tumor microenvironment, and the real-time reversible detection of hypoxia fluctuation in the tumor is an important index of poor prognosis such as tumor deterioration and metastasis. This example demonstrates the reversibility of the response of the complexes Mito-IrL3 and IrL4 to oxygen concentration. In 10. mu.M toluene solutions of the probe and the control probe, argon gas and an argon-oxygen mixed gas containing 21% by volume of oxygen gas were respectively circulated and measured for their intensities at their maximum phosphorescence emission peaks.
The experimental results are shown in FIG. 3, in which FIG. 3a shows the variation of phosphorescence intensity of complex Mito-IrL3 at 685nm at two concentrations of 0% and 21% of oxygen concentration, and in which FIG. 3b shows the variation of phosphorescence intensity of complex IrL4 at 685nm at two concentrations of 0% and 21% of oxygen concentration.
Within 5 cycles, Mito-IrL3 and IrL4 can realize the cycle of 'reduction-enhancement-reduction-enhancement' of the probe to the oxygen concentration, which shows that the probe complexes Mito-IrL3 and IrL4 can realize reversible phosphorescence detection and identification of the oxygen concentration.
Example 6 Effect of pH on the complexes Mito-IrL3 and IrL4
The complex should not be affected by pH change when used for intracellular imaging. Thus, the effect of pH on the complexes Mito-IrL3 and IrL4 was tested in PBS buffer (containing volume fraction 1% DMSO).
The experimental results are shown in FIG. 4, in which FIG. 4a shows the variation of the intensity of the maximum emission peak at 685nm of the complex Mito-IrL3 under different pH conditions, and FIG. 4b shows the variation of the intensity of the maximum emission peak at 685nm of the complex IrL4 under different pH conditions.
The maximum emission peak intensity of the two complexes at 685nm has no obvious change in the pH range of 4.5-11.5, so that the two complexes can still be used in an acidic environment, which is particularly important when the probe is applied to tumor hypoxia imaging. This shows that the two probes have good stability in physiological pH range and can be completely used for the imaging of biological systems.
EXAMPLE 7 photostability study of the complexes Mito-IrL3 and IrL4
The light stability is a very important factor for developing a fluorescent imaging probe, and compared with a small-molecule fluorescent compound, a transition metal phosphorescent complex has excellent photobleaching resistance. Photostability studies of the complexes Mito-IrL3 and IrL4 were performed in PBS buffer (containing a volume fraction of 1% DMSO).
The experimental results are shown in FIG. 5, in which FIG. 5a shows the change of the phosphorescence intensity of the complex Mito-IrL3 at the maximum emission wavelength under different illumination conditions, and FIG. 5b shows the change of the phosphorescence intensity of the complex IrL4 at the maximum emission wavelength under different illumination conditions.
When the complex Mito-IrL3 and IrL4 are continuously irradiated by a 180W xenon lamp, the phosphorescence intensity of the complex Mito-IrL3 and IrL4 at the maximum emission wavelength of 685nm has small change, so that the probe is proved to have good light stability, can be used for carrying out long-time radiography in cells or organisms without generating a light bleaching phenomenon, and provides a foundation for later imaging in biological systems.
EXAMPLE 8 determination of the singlet oxygen quantum yields of the complexes Mito-IrL3 and IrL4
(1) Preparing a DMSO solution of DPBF, and storing the solution with the concentration of 10mM as mother solution in a dark place; (2) a solution of probe RuL1 in acetonitrile was prepared at a concentration of 1 mM. The same concentration was prepared with the same solvent with reference to the standard Ru (bpy)32 +; (3) respectively taking a certain amount of DPBF mother liquor, and preparing a probe solution into a solution containing 10 mu M of DPBF and 10 mu M of probe concentration (wherein the absorbance of the probe at 450nm is between 0.1 and 0.3); (4) selecting a laser with the wavelength of 450nm according to the ultraviolet absorption wavelength of the probe, and measuring the ultraviolet absorption at a specific time; (5) the UV curve generated by the solution was recorded for a total of 100s of light and 10s of light, and plotted.
1, 3-diphenyl isobenzofuran (DPBF) is used as a high-efficiency singlet oxygen trapping agent, a continuous conjugated system exists in the molecular structure of the DPBF, and strong absorption is realized at 415 nm. The conjugated system is destroyed when DPBF reacts with singlet oxygen as follows, resulting in a significant decrease in the absorption at 415nm, which is shown below:
the singlet oxygen efficiency can be obtained according to the formula:
phi in the formulaΔ(1O2) Representing singlet oxygen yield, the superscripts "S" and "R" represent sample and standard, respectively. S represents the change of the ultraviolet absorption value at 415nm along with the extension of the illumination time by the ordinate of DPBF, and the abscissa of the DPBF represents the slope of the straight line obtained by detecting the time. F represents the light absorption amount of the sample solution after the light irradiation. F is represented by the formula F ═ 1-10-OD(OD value is an absorbance value of the sample at an excitation wavelength) was calculated. Standard substance Ru (bpy)3 2+The singlet yield in air-equilibrated acetonitrile was 0.56.
As shown in FIG. 6, FIG. 6a shows the time-dependent change of the absorption value of DPBF at 413nm caused by the illumination complex Mito-IrL3, and FIG. 6b shows the time-dependent change of the absorption value of DPBF at 413nm caused by the illumination complex IrL 4.
Through formula calculation, the singlet oxygen generation quantum yields of the complexes Mito-IrL3 and IrL4 are 0.76 and 0.81 respectively. Both singlet oxygen quantum yields are high, indicating that they have a high singlet oxygen generating capacity, which is very important in photodynamic therapy, and thus can be used as a photosensitizer for photodynamic therapy.
EXAMPLE 9 mitochondrial Co-localization experiment of the Complex Mito-IrL3
Cell culture
Human breast cancer cells MCF-7 cells were cultured in RPMI-1640 medium containing 10% Fetal Bovine Serum (FBS) and 100U/mL penicillin and 50U/mL streptomycin were added. The environment in the cell culture chamber was 37 ℃, 5% CO2, saturated humidity. Cell culture conditions with oxygen concentrations of 0% and 10% were constructed using anaerobic Gas-producing bags and microaerophilic Gas-producing bags (Mitsubishi Gas Chemical, Japan). To maintain the viability of the cells, all experimental cells were within two months of revival in liquid nitrogen.
MCF-7 cells were imaged after 2h incubation with 10. mu.M probe, excitation wavelength for probe imaging: 488 nm; emission wavelength range: 620 and 750 nm. MCF-7 cells were imaged after incubation with 1. mu.M Mito-Tracker Deep Red 633 for 10min, excitation wavelength of co-stain contrast: 633 nm; emission wavelength range: 670-750 nm.
The results are shown in FIG. 7, in which FIG. 7a shows the distribution of the complex Mito-IrL3 in mitochondria and FIG. 7b shows the distribution of the intensity of Mito-IrL3 and Mito-Tracker Deep Red 633 in cells. The results show that the complex Mito-IrL3 can enter cells quickly, and meanwhile, the triphenylphosphine targeting group introduced into the structure of the complex makes the complex possibly have the capacity of targeting mitochondria, so that the distribution of the complex Mito-IrL3 in the cells is studied next, and the comparison with IrL4 is carried out. The subcellular organelle co-localization study was performed using a laser confocal fluorescence microscope. As can be seen from FIG. 7, the phosphorescence of the complex Mito-IrL3 is well overlapped with the fluorescence of the commercial mitochondrial dye Mito-Tracker Deep Red 633, the overlapping degree is 0.85, and the complex Mito-IrL3 is mainly localized in mitochondria after entering cells and has better mitochondrial targeting performance.
Meanwhile, the degree of superposition of phosphorescence of the complex IrL4 and fluorescence of a commercial mitochondrial dye is low (Pearson co-localization coefficient is 0.47), which indicates that the complex has no specific distribution in mitochondria. The above experiments show that the introduction of triphenylphosphine groups into the complex Mito-IrL3 does contribute to its selective enrichment in mitochondria.
Example 10 study of hypoxic response in Mito-IrL3 and IrL4 cells with the Complex
The invention tests the response of the probe to hypoxia in cells cultured in vitro. MCF-7 cells were co-cultured with the complexes Mito-IrL3 and IrL4, respectively, at different oxygen concentrations (0%, 10% and 21%), and then the phosphorescence image of the cells was observed.
The results of the experiment are shown in FIG. 8, and show that for the complex Mito-IrL3, the probe signal detected from the cell is extremely weak at atmospheric oxygen concentration (21%), whereas when the oxygen concentration is reduced to 0%, a strong probe signal can be detected from the cell. Similar results were obtained when the complex IrL4 was co-cultured with MCF-7 cells at different partial pressures and imaged. Therefore, the complexes Mito-IrL3 and IrL4 have obvious hypoxic selectivity on cell imaging, and have potential application in the hypoxic detection of tumors.
EXAMPLE 11 study of photodynamic therapeutic Activity of the complexes Mito-IrL3 and IrL4 under hypoxic conditions
The invention researches the photodynamic therapeutic activity of the complexes Mito-IrL3 and IrL4 under the hypoxic condition. MCF-7 cells were seeded in 96-well plates (approximately 5000 cells per well) at 37 ℃ in 5% CO2Culturing for 24h under the condition, and after the cells are attached, culturing for 2h by changing to RPMI-1640 culture solution containing different concentrations of complex Mito-IrL3(5 mu M and 10 mu M) or IrL4(1 mu M and 5 mu M). After the incubation is finished, the fresh culture medium is replaced, and the cells are placed under the hypoxic condition for culture for 1h and then are illuminated. Wherein the illumination adopts 450nm laser (from Ningbo laser technology Limited) illumination (30J/cm)2300 s). And (3) calculating the cell survival rate under each condition by adopting an MTT method at 0h, 1h, 2h and 4h after the illumination by taking the illumination end as 0 point, wherein the toxic activity experiment needs to be repeated three times.
The experimental results are shown in fig. 9, in which fig. 9a shows the change of the survival rate of the MCF-7 cells with time after the co-incubation of the complex Mito-IrL3 with the MCF-7 cells under the normoxic and hypoxic conditions, and fig. 9b shows the change of the survival rate of the MCF-7 cells with time after the co-incubation of the complex IrL4 with the MCF-7 cells under the normoxic and hypoxic conditions.
Under the condition of normal oxygen, the complexes Mito-IrL3 and IrL4 show better killing effect on MCF-7 cells under the irradiation of light. For the complex Mito-IrL3 and MCF-7 cells which are incubated for 4 hours, the obvious apoptosis of the cells is not caused, after illumination, the cell survival rates of 5 mu M and 10 mu M drug incubation groups are respectively reduced to 8.9 percent and 9.6 percent under normal oxygen, the cell survival rates of hypoxic groups are reduced to 28.8 percent and 13.6 percent, and the survival rates of four groups of cells are about 10 percent after continuous culture for 4 hours. In the case of the complex IrL4, the cell survival rate is reduced to 31.8% and 14.8% under the condition of illumination of 1 μ M and 5 μ M drugs under normal oxygen, the cell survival rate is divided into 104.1% and 33.4% under hypoxic, and the cell survival rate is further reduced after the culture is continued for 4 hours. As can be seen from the control of the two groups, the complex Mito-IrL3 shows higher photodynamic treatment efficiency on MCF-7 cells than IrL4 under normal oxygen and anaerobic oxygen.
Example 12 study of Mito-IrL3 and IrL4 complexes to induce changes in mitochondrial Membrane potential
In the process of photodynamic therapy, the type I mechanism mainly occurs in various biological membranes of cells, for example, the type I mechanism generates hydrogen extraction reaction with unsaturated phospholipid molecules and further generates lipid peroxide after the reaction with oxygen, and the lipid peroxide reaction causes the structural integrity of the membrane to be damaged and the ion permeability to be increased. Since the complex Mito-IrL3 is mainly localized to mitochondria, the effect of Mito-IrL3 and IrL4 on mitochondrial membrane potential (. DELTA.. psi.m) under light in normoxic and hypoxic conditions was studied. Mitochondrial membrane potential change studies were performed using the JC-1 mitochondrial membrane potential kit. JC-1 is an ideal fluorescent probe widely used for detecting mitochondrial membrane potential, and can detect the mitochondrial membrane potential of cells, tissues or purification. When the mitochondrial membrane potential is high, JC-1 is accumulated in a matrix (matrix) of mitochondria to form a polymer (J-aggregates), and red fluorescence can be generated; at a low mitochondrial membrane potential, JC-1 cannot be aggregated in the matrix of mitochondria, and at the moment, JC-1 is a monomer (monomer) and can generate green fluorescence, so that the change of the mitochondrial membrane potential can be conveniently detected through the conversion of fluorescence color.
The experimental result is shown in fig. 10, in which fig. 10a is a confocal imaging diagram of the fluorescence change of JC-1 after the complex Mito-IrL3 is incubated with JC-1 under the conditions of normal oxygen and hypoxic oxygen, and fig. 10b is a confocal imaging diagram of the fluorescence change of JC-1 after the complex IrL4 is incubated with JC-1 under the conditions of normal oxygen and hypoxic oxygen.
The two complexes and MCF-7 cells are incubated for 4 hours in the dark, and the cells mainly emit red fluorescence, which proves that Mito-IrL3 and IrL4 do not cause the decrease of mitochondrial membrane potential in the dark at the concentration of 5 mu M. In addition, under the condition of normal oxygen illumination, the red fluorescence in cells incubated by the two complexes is mainly converted into green fluorescence, so that the fact that the light illumination under normal oxygen affects mitochondria is proved to cause the reduction of membrane potential. In the hypoxic light experiment, the cell incubated with Mito-IrL3 mainly becomes green fluorescence, while IrL4 still mainly becomes red.
The decrease in mitochondrial membrane potential is a marker event in the early stages of apoptosis. The reduction of the mitochondrial membrane potential of the cell can be easily detected by the conversion of JC-1 from red fluorescence to green fluorescence, and the conversion of JC-1 from red fluorescence to green fluorescence can also be used as an early detection index of apoptosis. From the above results, the complex Mito-IrL3 can obviously cause the decrease of mitochondrial membrane potential under the condition of hypoxia, which is consistent with that Mito-IrL3 hypoxia photodynamic therapy activity in the photodynamic therapy experiment is stronger than that of IrL 4.
Claims (10)
1. A cyclometalated iridium complex is characterized by having the following structure:
2. The cyclometalated iridium complex of claim 1 wherein the complex is bound to an anion.
3. The cyclometalated iridium complex of claim 1 wherein the anion is selected from the group consisting of Cl-、Br-，I-，NO3 -Or PF6 -。
4. Use of a cyclometalated iridium complex according to any one of claims 1 to 3 for the preparation of a photosensitizer.
5. Use according to claim 4, characterized in that the cyclometalated iridium complex is a photosensitizer associated with photodynamic therapy.
6. The use according to claim 4, wherein the cyclometalated iridium complex is used as a photosensitizer for confocal imaging of cells.
7. The use according to claim 4, characterized in that the cyclometalated iridium complex is used for preparing antitumor drugs.
8. Use according to claim 7, characterized in that said tumors are selected from the group consisting of melanoma, breast cancer, cervical cancer, gastric cancer, lung cancer or liver cancer.
9. The use according to claim 4, characterized in that the cyclometalated iridium complex is used for preparing a reagent for detecting oxygen in living cells.
10. The use according to claim 9, wherein the cyclometalated iridium complex is reversibly phosphoresced for oxygen concentration detection.
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