Lysosome-targeted iridium complex and preparation method and application thereof

Abstract

The application provides a kind of lysosome-targeted iridium complex and its preparation method and application.Iridium complex cation and coordination anion comprising the structure shown in formula I or formula II.The metal iridium complex prepared by the application has novel structure, lysosome targeting property and phosphorescence property, can not only realize phosphorescence imaging of cell and tissue, but also can be used as photosensitizer to produce reactive oxygen under light, for photodynamic therapy of tumor.In weak acid condition, the phosphorescence of the complex is significantly enhanced, and the ability to produce singlet oxygen and catalyze NADH is also increased, so it has more effective killing ability to lysosome and tumor.The preparation method of the application has the advantages of simple preparation process, high yield and good repeatability.The metal iridium complex prepared by the application can be used for biological imaging and photodynamic therapy of tumor.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of coordination chemistry, biology, and medicine, and specifically relates to a lysosome-targeted iridium complex, its preparation method, and its application in bioimaging and tumor photodynamic therapy. Background Technology

[0002] In recent years, metal complexes have attracted increasing attention as anti-tumor drugs. Cisplatin and its derivatives, in particular, have been widely used clinically to treat various types of cancer and play an important role in combination regimens. However, the drug tolerance and side effects of platinum (Pt)-based chemotherapy drugs limit their application. Therefore, the development of novel metal drugs is crucial. Among various metal drugs, cyclic metallized iridium complexes have emerged as promising candidates for cancer treatment due to their tunable structure and redox potential, large Stokes shift, significant photostability, long excited-state lifetime, and excellent color tunability. With these advantages, cyclic metallized iridium complexes exhibit unique benefits in optical bioimaging, targeting subcellular organelles, chemotherapy, and photodynamic therapy.

[0003] Photodynamic therapy targeting subcellular organelles is a promising approach. Lysosomes play crucial roles in various physiological and signal transduction processes, such as intracellular transport, protein degradation, endocytosis, and cell death, making them a promising target. When lysosomes are disrupted, cathepsins and other hydrolytic enzymes within them are released into the cytoplasm. These enzymes can trigger programmed cell death by cleaving various substrates, such as caspases and several members of the Bcl-2 protein family. Photosensitizers targeting lysosomes can further enhance therapeutic efficacy while reducing photosensitizer dosage, minimizing side effects, and avoiding drug tolerance.

[0004] Although some photosensitizers targeting lysosomes have been reported in the literature, the hypoxic nature of tumor tissues can severely reduce the photodynamic therapeutic efficacy of type II photosensitizers, which are highly oxygen-dependent. There is an urgent need to develop new methods to overcome the limitations of tumor hypoxia. Furthermore, compared to normal tissues and blood (pH approximately 7.4), the tumor microenvironment is more acidic (pH 6.5-6.8), with lysosomes having a pH range of 4.5-5.5. Therefore, developing photosensitizers that target lysosomes and are activated by acidic pH conditions could potentially significantly increase the efficacy of tumor therapy. Summary of the Invention

[0005] In view of this, the present invention provides a metallic iridium complex, its preparation method and application.

[0006] The metal iridium complexes provided by the present invention include iridium complex cations and coordinating anions with the structures shown in Formula I or Formula II;

[0007]

[0008] The iridium complex cation shown in Formula I can be protonated to the structure shown in Formula II under low pH conditions;

[0009] The coordinating anion includes Cl. - ,Br - I - NO3 - and PF6 - At least one of them.

[0010] Preferably, the coordinating anion is PF6. - .

[0011] The present invention also provides a method for preparing the above-mentioned metallic iridium complex.

[0012] The method for preparing the metallic iridium complex provided by this invention includes the following steps:

[0013] 1) Dissolve 1,10-phenanthroline-5,6-dione, p-hydroxybenzaldehyde and ammonium acetate in an organic solvent and react to prepare intermediate 1. The structural formula of intermediate 1 is shown in Formula I-1.

[0014]

[0015] Formula I-1

[0016] 2) Intermediate 1 and the iridium complex precursor shown in Formula I-2 are subjected to a coordination reaction in a mixed solvent of dichloromethane and methanol to obtain a complex containing the iridium cation shown in Formula I and Cl. - It is a metal iridium complex that coordinates anions;

[0017]

[0018] Formula I-2

[0019] 3) The above-mentioned iridium complex cation containing formula I, Cl - The iridium complex with the coordinating anion undergoes an ion exchange reaction to yield Br. - I - NO3 - or PF6 - A metal iridium complex with a coordinating anion and structural formula I as a cation;

[0020] 4) The obtained metallic iridium complex is protonated under acidic conditions to obtain a metallic iridium complex containing the iridium complex cation shown in Formula II.

[0021] In step 1) of the above method, the molar ratio of 1,10-phenanthroline-5,6-dione, p-hydroxybenzaldehyde and ammonium acetate is 1:1:10.

[0022] The organic solvent is glacial acetic acid.

[0023] The reaction temperature can be 80-120℃, specifically 120℃, and the time can be 12-24h, specifically 24h.

[0024] In step 2) of the above method, the ratio of the number of moles of intermediate 1 to the sum of the number of moles of intermediate 1 and iridium complex precursor is 0.2-0.8:1; specifically, it can be 0.5:1.

[0025] The coordination reaction is carried out under reflux, and the time of the coordination reaction can be 12-24 hours, specifically 24 hours.

[0026] The ion exchange reaction is to remove Cl... - Replace with Br - I - NO3 - or PF6 - Reagents used for ion exchange reactions include NaBr, NaI, NaNO3, or ammonium hexafluorophosphate;

[0027] The ion exchange reaction can be carried out at room temperature, and the reaction time can be 1-4 hours, specifically 1 hour.

[0028] The iridium complex has lysosomal targeting properties.

[0029] The iridium complex can generate reactive oxygen species under light, and its ability to generate reactive oxygen species is even stronger under acidic conditions, thus exhibiting enhanced photodynamic effects.

[0030] The iridium complex exhibits photocatalytic redox capabilities independent of oxygen concentration, and these capabilities are even stronger under acidic conditions.

[0031] The application of the above-mentioned iridium metal complexes in the preparation of fluorescence imaging reagents also falls within the scope of protection of this invention.

[0032] The present invention also provides a photosensitizer comprising the above-mentioned metal iridium complex.

[0033] The use of the iridium complex and / or photosensitizer in the preparation of antitumor drugs; preferably, the antitumor drugs include drugs suitable for photodynamic therapy.

[0034] The tumor cells for which the anti-tumor drugs are applicable include breast cancer 4T1 cells and breast cancer EMT6 cells.

[0035] The antitumor drug targets the lysosomes of tumor cells.

[0036] The iridium metal complex, as a photosensitizer, exhibits pH-responsive phosphorescence properties. This photosensitizer demonstrates low in vitro cellular dark toxicity and high phototoxicity.

[0037] The iridium complex prepared in this invention has a novel structure and exhibits lysosomal targeting and phosphorescence properties. It can not only achieve phosphorescent imaging of cells and tissues but also act as a photosensitizer, generating reactive oxygen species under light irradiation for photodynamic therapy of tumors. Under weakly acidic conditions, the phosphorescence of this complex is significantly enhanced, and its ability to generate singlet oxygen and catalyze NADH is also increased, thus demonstrating a more effective killing effect on lysosomes and tumors. The preparation method of this invention has advantages such as simple preparation process, high yield, and good reproducibility. The iridium complex prepared in this invention can be used for bioimaging and photodynamic therapy of tumors. Attached Figure Description

[0038] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0039] In the attached diagram:

[0040] Figure 1 This is a synthetic route diagram of the metallic iridium complex in Example 1 of the present invention.

[0041] Figure 2 This is a high-resolution mass spectrum of the iridium complex in Example 1 of the present invention.

[0042] Figure 3 This is the hydrogen spectrum of the iridium complex in Example 1 of the present invention.

[0043] Figure 4 The images show the ultraviolet and phosphorescence spectra of the iridium complex in Example 2 of this invention.

[0044] Figure 5 This is a laser confocal microscopy image of the iridium complex in mouse breast cancer cells 4T1 after co-staining with mitochondrial probes MTR and LTR, respectively, in Example 3 of the present invention.

[0045] Figure 6 This is a diagram showing the effect of reactive oxygen generation of the iridium complex in two different pH aqueous solutions in Example 4 of the present invention.

[0046] Figure 7 This is a test graph showing the photocatalytic oxidation of NADH by the iridium complex in Example 5 of the present invention in two aqueous solutions with different pH values.

[0047] Figure 8 This is a small animal in vivo fluorescence imaging image of the iridium complex against mouse tumors in Example 6 of the present invention.

[0048] Figure 9 This is a real-time monitoring image of the destruction of lysosomes by the iridium complex under light in Example 7 of the present invention.

[0049] Figure 10 This is a diagram illustrating the tumor-killing effect of the iridium complex at the cellular level in Example 8 of the present invention.

[0050] Figure 11 This is a diagram illustrating the tumor-killing effect of the iridium complex in Example 9 of the present invention at the in vivo level. Detailed Implementation

[0051] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0052] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0053] Example 1

[0054] Preparation and characterization of lysosome-targeted iridium complexes, synthetic steps as follows: Figure 1 As shown. (1) Synthesis of intermediate 1. 1,10-phenanthroline-5,6-dione (1.05 g), p-hydroxybenzaldehyde (0.61 g) and ammonium acetate (3.85 g) were added to the reaction vessel, and 60 mL of glacial acetic acid was added. Under argon protection, the mixture was stirred at 120 °C for 24 hours. After cooling to room temperature, the pH was adjusted to neutral with concentrated ammonia. A large amount of white solid appeared. The mixture was filtered, and the precipitate was collected to obtain intermediate 1. (2) Synthesis of the target product. Intermediate 1 (312 mg) and the iridium complex precursor (540 mg) were added to a CH2Cl2 / MeOH (1 / 1 v / v) mixed solvent, and refluxed for 24 hours under argon protection. After the reaction was completed, the mixture was rotary evaporated to dryness, dissolved in a small amount of dichloromethane, and ammonium hexafluorophosphate was added. The mixture was reacted at room temperature for 1 hour, and the target product, the metal iridium complex, was obtained by column chromatography. (3) Characterization of the metal iridium complex. The obtained complex was dissolved in acetonitrile and characterized by electrospray ionization mass spectrometry. The results are as follows: Figure 2 As shown. (4) The obtained iridium complex was dissolved in DMSO-d6, and the proton spectrum was measured by nuclear magnetic resonance, as shown. Figure 3 As shown.

[0055] Example 2

[0056] The UV absorption and fluorescence emission properties of lysosome-targeted iridium complexes. For example... Figure 4 As shown, the iridium complex prepared in Example 1 exhibits a moderate absorption peak at 279 nm and a shoulder peak around 380 nm, which are typical absorption peaks caused by MLCT transitions. This iridium complex also emits light in the 470-830 nm range, with an emission peak at 605 nm.

[0057] Example 3

[0058] Live-cell optical imaging of lysosome-targeted iridium complexes. Mouse breast cancer 4T1 cells in logarithmic growth phase were separated into two 35mm confocal culture dishes and grown adherently in 1640 medium containing 10% fetal bovine serum at 37°C for 24 hours. Then, 30 μM of the iridium complex prepared in Example 1 was added to each dish, and the cells were incubated for 4 hours. After washing with PBS, mitochondrial red fluorescent probe MTR and lysosomal red fluorescent probe LTR were added to the two confocal dishes, respectively, and incubated for 20 minutes. Confocal microscopy was then performed immediately. The experimental results are shown below. Figure 5 As shown, the iridium complex only partially overlaps with the mitochondrial probe MTR, but almost completely overlaps with the lysosomal probe LTR, indicating that the iridium complex is located in the lysosome of the cell, demonstrating its lysosomal targeting.

[0059] Example 4

[0060] Determination of reactive oxygen species (ROS) generation by iridium metal complexes under light irradiation. To investigate the ability of iridium metal complexes to generate ROS under light irradiation, this invention utilizes the ROS probe 9,10-anthratridiyl-bis(methylene)dicarboxylic acid (ABDA) to detect ROS generation. Figure 6 As shown, when the iridium complex coexists with ABDA, the absorption peak of ABDA at 378 nm gradually decreases with increasing light exposure time, indicating that the iridium complex can generate reactive oxygen species under light conditions. Simultaneously, this invention selected two buffer solutions with different pH values, such as... Figure 6 As shown, under low pH conditions (pH=4.5), the absorption peak of ABDA decreases more significantly over time, indicating that the iridium complex has a stronger ability to generate reactive oxygen species under acidic conditions and has an enhanced photodynamic effect.

[0061] Example 5

[0062] Determination of photocatalytic oxidation of NADH by iridium complexes under illumination. Since tumor tissues are hypoxic, the efficacy of oxygen concentration-dependent photodynamic therapy is inevitably weakened. In recent years, oxygen concentration-independent photocatalytic redox mechanisms have been considered a novel and highly effective strategy to overcome tumor hypoxia limitations. The iridium complexes of this invention can undergo photocatalytic redox reactions with the mitochondrial coenzyme NADH, such as... Figure 7 As shown, the characteristic absorption peak of NADH at 338 nm gradually decreases over time, indicating that this iridium complex can undergo photocatalytic redox reactions. Furthermore, under low pH conditions (pH = 4.5), the decrease in the absorption peak of NADH over time is more pronounced, indicating that this iridium complex has stronger photocatalytic redox capabilities under acidic conditions.

[0063] Example 6

[0064] Small animal in vivo fluorescence imaging of iridium complexes for mouse tumors. A mouse 4T1 tumor model was established. Two tumor-bearing mice with tumor volumes of approximately 100 mm³ were selected. One mouse served as a control, receiving only saline injection, while the other served as the experimental group, receiving 25 μL of iridium complex intratumorally. Imaging was performed using a small animal in vivo fluorescence imaging system. Figure 8 As shown, the tumor sites in the control group showed almost no fluorescence, while obvious fluorescence signals were observed in the tumor sites of the experimental group mice, indicating that this iridium complex can be used for in vivo fluorescence imaging in mice.

[0065] Example 7

[0066] The iridium complex disrupts lysosomes in tumor cells. Since this iridium complex targets lysosomes and in vitro experiments showed that it generates reactive oxygen species under light conditions, we further investigated its lysosomal killing effect on tumor cells using acridine orange as an indicator. We incubated the iridium complex in 4T1 cells for 3 hours, then added acridine orange and incubated for half an hour. After removing the culture medium, the cells were washed three times with PBS and imaged using laser confocal microscopy. Fluorescence in the green and red channels was collected separately. The results are shown below. Figure 9 As shown, in the absence of laser irradiation (0 min), green fluorescence was almost distributed throughout the entire cell, while red fluorescence was only distributed in the lysosomes. Subsequently, the cells were irradiated using a laser confocal microscope, and fluorescence signals were collected every 40 seconds until the experiment ended after 200 seconds. Real-time dynamic monitoring images show that after laser irradiation, the fluorescence signal of some lysosomes in tumor cells disappeared and diffused, indicating that these lysosomes were destroyed. This indirectly proves that the iridium complex can kill lysosomes in tumor cells, thereby achieving better therapeutic effects.

[0067] Example 8

[0068] The killing effect of iridium complexes on tumor cells. To investigate whether this iridium complex can be used as a photosensitizer to kill tumor cells, this invention uses a cell viability assay kit to demonstrate that the iridium complex can kill cells. Figure 10As shown, 4T1 cells were treated with the following different methods: 1) control group; 2) light-only group; 3) complex only group; 4) iridium complex + light-only group; 5) iridium complex + light-only group + inhibitor group. The results showed that only in the iridium complex + light-only group did the green fluorescence of calcein disappear, while the red fluorescence of propidium iodide appeared, indicating that programmed cell death had occurred. The other groups only showed fluorescence of calcein, without the fluorescence of propidium iodide, indicating that the cells were viable. This experiment demonstrates that the iridium complex can kill cells under light irradiation, exhibiting good photodynamic therapy efficacy.

[0069] Example 9

[0070] In vivo fluorescence imaging of mouse tumors using a mouse tumor model of iridium complexes. To investigate the tumor-killing effect of this iridium complex, this invention evaluated its tumor-killing efficacy by constructing a tumor-bearing mouse model. First, 20 female Balb / c mice were randomly selected, and each mouse was subcutaneously injected with one million 4T1 tumor cells. After approximately 7 days, the tumor volume reached about 100 cubic millimeters for further experiments. These mice were randomly divided into four groups: 1) control group; 2) light-only group; 3) complex-only group; and 4) complex + light-only group. For the control group, 25 μL of physiological saline was injected into the tumor; for the light-only group, the tumor site was irradiated with a 390 nm LED lamp for 1 hour; for the complex group, 25 μL of iridium complex (5 mg / kg) was injected into the tumor; and for the complex + light-only group, the iridium complex was injected into the tumor followed by light treatment. Results are as follows... Figure 11 As shown, only the complex + light irradiation group exhibited significant tumor inhibition; tumors almost completely disappeared in 2 out of 5 mice, and tumors were also significantly inhibited in the other 3 mice. These results indicate that this iridium complex possesses excellent photodynamic tumor-killing ability and holds promise for further clinical applications.

[0071] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of a metal iridium complex in the preparation of a fluorescent imaging reagent, wherein the fluorescent imaging reagent is used for optical imaging of breast cancer 4T1 cells, and the metal iridium complex targets lysosomes in the cells; the structural formula of the metal iridium complex is as follows: 。

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