Ruthenium-based photosensitizer, preparation method thereof and application of ruthenium-based photosensitizer in tumor photodynamic therapy
By designing a photoresponsive ruthenium-based photosensitizer, the problems of insufficient stability, water solubility and targeting of existing photosensitizers in photodynamic therapy are solved, and effective killing and therapeutic effects on triple-negative breast cancer cells in photodynamic therapy are achieved.
Patent Information
- Application Number
- CN202510066856.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-16
- Publication Date
- 2025-05-27
AI Technical Summary
Existing photosensitizers are insufficient in stability, water-soluble and targeting in photodynamic therapy, and may cause side effects, making it difficult to effectively treat triple-negative breast cancer.
A photoresponsive ruthenium-based photosensitizer was designed. Its cationic structure was formed by combining with anionic chloride ions. It adopts a simple preparation method and a synthesis route with high reaction yield. It can generate singlet oxygen under light and significantly induce the death of triple-negative breast cancer cells.
The ruthenium-based photosensitizer exhibits excellent anti-tumor properties in photodynamic therapy, can effectively kill triple-negative breast cancer cells, reduce the risk of recurrence, and is simple in preparation and low in cost, suitable for large-scale production and application.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biomedicine, and relates to a ruthenium-based photosensitizer, a preparation method thereof, and an application thereof in tumor photodynamic therapy. Background Art
[0002] The incidence and mortality of cancer are increasing rapidly worldwide. Breast cancer is the most common malignant tumor in women. Early breast cancer is considered curable. Surgical resection, radiotherapy, chemotherapy and endocrine therapy are the main treatments for breast cancer, but metastasis and drug resistance still make treatment difficult, and the prognosis of advanced breast cancer is poor. Triple Negative Breast Cancer (TNBC) is the most aggressive subtype of breast cancer. It has a high risk of metastasis and recurrence, is significantly resistant to chemotherapy drugs, and often has a worse prognosis than other types. TNBC is negative for estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2), resulting in poor results of traditional endocrine therapy and targeted therapy. Therefore, research on new treatment methods is of great significance to improve the treatment effect and prognosis of TNBC.
[0003] Photodynamic therapy (PDT) is a new technology that applies the principles of photochemistry, photophysics and photobiology to the diagnosis and treatment of diseases. The basic principle is to induce photosensitizers in the lesion tissue to produce reactive oxygen species (ROS) through light, and then destroy the diseased cells through oxidative damage. PDT is a non-invasive treatment method that can be used in the treatment of cancer. Compared with traditional cancer treatment methods, PDT has the characteristics of targeting, minimally invasive / non-invasive, few side effects, and repeatability. Studies have shown that PDT can improve the comprehensive treatment effect of cancer. Combining PDT after surgery can effectively reduce tumor tissue residues, reduce the risk of recurrence, and help improve patients' survival and quality of life. However, the photosensitizers currently used in clinical practice still have defects. It is difficult to have stability, water solubility and targeting. It may also cause more obvious side effects. New photosensitizers need to be developed.
[0004] Ruthenium, a hard and brittle light grey multivalent rare metal, has attracted much attention due to its unique chemical properties. Its easy formation of hexacoordinated complexes provides rich possibilities for scientific research. Among the many properties of ruthenium complexes, good thermodynamic stability, rich photochemical and photophysical information, and long excited state lifetime are particularly eye-catching. These characteristics just meet the development needs of photosensitizers, making ruthenium-based photosensitizers show great potential in the field of photodynamic therapy (PDT). As an emerging treatment method, photodynamic therapy is highly praised for its non-invasive and efficient characteristics, especially in tumor treatment. Ruthenium-based photosensitizers, as the leaders in this field, have naturally become a hot topic of research. However, despite many related studies at home and abroad, unfortunately, the PDT effect of ruthenium-based photosensitizers is not ideal at present, and only a few can successfully enter clinical trials. Faced with this challenge, scientists have not stopped. They know that adjusting and modifying the ligands and continuously optimizing the structure of ruthenium complexes are the key to improving the PDT effect of ruthenium-based photosensitizers. This basic approach not only tests the wisdom and patience of scientists, but also carries the desire and expectation of countless patients for a healthy life. It is in this context that the present invention came into being. By adjusting and modifying the ligand and continuously optimizing the structure of the ruthenium complex, it is a basic approach to improve the PDT effect of the ruthenium complex. Based on the existing research results, it has designed a new ruthenium-based photosensitizer through in-depth exploration and experiments. Summary of the invention
[0005] The present invention provides a ruthenium-based photosensitizer and a preparation method thereof and application thereof in tumor photodynamic therapy.
[0006] The present invention is achieved through the following technical solutions:
[0007] A ruthenium-based photosensitizer with light responsiveness, the cationic structural formula of which is as follows:
[0008]
[0009] It is further described in the present invention that the cation can be further combined with the anion chloride ion to form a ruthenium-based photosensitizer.
[0010] The present invention also provides a method for preparing a ruthenium-based photosensitizer with light responsiveness, comprising the following steps:
[0011] (1) ruthenium trichloride trihydrate, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine and anhydrous lithium chloride are mixed in N,N-dimethylformamide, heated to 150° C. and refluxed for 8 hours. After the reaction is completed, the reaction solution is cooled to room temperature, and pure water is added to the reaction solution until no solid is precipitated. The solid is collected by suction filtration, washed with water, and dried to obtain a crude product of a ruthenium-based photosensitizer;
[0012] (2) Purify the sample obtained in step (1) by column chromatography to obtain the ruthenium-based photosensitizer as described in claim 1.
[0013] Further illustrate that in step (1), the molar ratio of ruthenium(III) chloride trihydrate, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine, and lithium chloride anhydrous is 5:15:23.
[0014] Further illustrate that in step (2), the adsorbent used in the column chromatography is neutral alumina, and the eluent is a mixed solution of dichloromethane and ethanol with a volume ratio of 9:1.
[0015] The present invention also provides an application of a ruthenium-based photosensitizer with photosensitivity in the photodynamic therapy of triple-negative breast cancer.
[0016] The synthetic route of the ruthenium-based photosensitizer with photosensitivity of the present invention is shown in the appendix Figure 1 .
[0017] Compared with the prior art, the present invention has the following advantages:
[0018] The ruthenium-based photosensitizer described in the present invention shows unique advantages in the field of photodynamic therapy. (1) The preparation method of this ruthenium-based photosensitizer is relatively simple, without complex synthesis steps and harsh reaction conditions. This reduces the production cost and improves the production efficiency, making the large-scale production and application of this complex possible. (2) During the preparation process, the reaction yield of this ruthenium-based photosensitizer is relatively high. This means that under the same reaction conditions, more target products can be obtained, thereby improving the utilization rate of raw materials and the purity of products. This is very beneficial for the subsequent separation and purification and drug preparation processes. (3) This ruthenium-based photosensitizer can generate singlet oxygen ( 1 O 2 ) under light irradiation. This is a highly reactive oxygen molecule with strong oxidation ability. During the photodynamic therapy process, singlet oxygen can react with biomolecules in cells, resulting in cell damage and death. Therefore, this ruthenium-based photosensitizer can effectively play the role of photodynamic therapy. (4) Triple-negative breast cancer is a type of breast cancer with a relatively high degree of malignancy and is difficult to treat. Traditional treatment methods such as surgery, radiotherapy, and chemotherapy can, to a certain extent, control the development of the disease, but are often accompanied by relatively large side effects and recurrence risks. As an emerging treatment method, photodynamic therapy has the advantages of non-invasiveness, high efficiency, and low toxicity, providing a new option for the treatment of triple-negative breast cancer.
[0019] This ruthenium-based photosensitizer can generate singlet oxygen under light irradiation, which has a killing effect on triple-negative breast cancer cells. At the same time, due to its simple preparation method and high reaction yield, this complex has broad application prospects in the field of photodynamic therapy.
[0020] In summary, the ruthenium-based photosensitizer described in the present invention shows unique advantages in aspects such as preparation method, reaction yield, generation of singlet oxygen under light irradiation, and application in photodynamic therapy of triple-negative breast cancer. These advantages make this complex have broad application prospects and important research value in the field of photodynamic therapy. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 It is a synthetic route diagram of the ruthenium-based photosensitizer of the present invention.
[0022] Figure 2 It is a mass spectrometry result diagram of the ruthenium-based photosensitizer of the present invention.
[0023] Figure 3 It is an ultraviolet-visible absorption spectrum diagram of the ruthenium-based photosensitizer of the present invention.
[0024] Figure 4 It is the change of the ultraviolet-visible absorption spectrum of the singlet oxygen probe DPBF caused by the ruthenium-based photosensitizer of the present invention under 635 nm laser irradiation.
[0025] Figure 5 It is the toxicity experiment of the ruthenium-based photosensitizer of the present invention on 4T1 cells.
[0026] Figure 6 It is the live / dead cell staining diagram of 4T1 cells treated with the ruthenium-based photosensitizer of the present invention.
[0027] Figure 7 It is the flow cytometry diagram of 4T1 cells treated with the ruthenium-based photosensitizer of the present invention. DETAILED DESCRIPTION OF THE INVENTION
[0028] The following further illustrates the specific implementation manners of the present invention in conjunction with embodiments.
[0029] Example 1: Preparation of Ruthenium-Based Photosensitizer
[0030] Weigh ruthenium(III) chloride trihydrate (52.29 mg, 0.2 mmol), 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine (186.21 mg, 0.6 mmol), and anhydrous lithium chloride (39 mg, 0.92 mmol) and place them in a round-bottom flask. Add 3 mL of N,N-dimethylformamide, heat to 150 °C, and reflux for 8 h. After the reaction is completed, let it cool to room temperature. Add 30 mL of pure water to the reaction solution, and a precipitate will form. Collect the solid by suction filtration, wash it with water, and dry it to obtain the crude ruthenium-based photosensitizer. Purify the crude product by column chromatography, using neutral alumina as the adsorbent and a mixture of dichloromethane and ethanol with a volume ratio of 9:1 as the eluent. After drying the purified product, the finally obtained ruthenium-based photosensitizer is a red-black solid.
[0031] The technical synthesis route of the finally obtained ruthenium-based photosensitizer is as Figure 1 shown.
[0032] The characterization data of the finally obtained ruthenium-based photosensitizer are as follows: 1 H NMR (DMSO-d 6 , 800 MHz) δ: 8.97 (d, 1H), 8.87 (d, 1H), 8.85 - 8.84 (m, 1H), 8.74 (d, 1H), 8.64 (d, 1H), 8.53 (d, 1H), 8.38 (q, 2H), 8.33 - 8.28 (m, 2H), 8.19 (d, 1H), 8.00 (t, 1H), 7.90 (t, 1H), 7.81 - 7.78 (m, 2H), 7.71 (t, 3H), 7.64 (t, 3H), 7.60 - 7.54 (m, 3H), 7.49 (t, 3H), 7.45 (t, 4H), 7.36 - 7.33 (m, 4H), 7.18 (d, 1H), 7.13 (d, 1H), 7.11 - 7.09 (m, 4H), 7.00 (d, 1H). MS: m / z 516.13 [M - 2Cl - 2+ .
[0033] The mass spectrometry result graph of the finally obtained ruthenium-based photosensitizer is as Figure 2 shown.
[0034] Example 2: Ultraviolet-visible absorption spectrum
[0035] Zero and calibrate the baseline of the ultraviolet-visible spectrophotometer with absolute ethanol in advance. Take a small amount of the ruthenium-based photosensitizer from Example 1, dissolve it in 3 mL of absolute ethanol, and place it in a cuvette. Add the same volume of absolute ethanol to the cuvette used as a control, and measure the absorbance of the sample at 200 - 800 nm on the machine. The results are as Figure 3 shown.
[0036] Example 3: Detection of Singlet Oxygen Generation in Vitro
[0037] Prepare a DMSO solution of DPBF as a stock solution for storage at a concentration of 10 mM. Take 8 μL of the DPBF stock solution, dilute it to 8 mL with absolute ethanol, add the above ruthenium-based photosensitizer and dissolve and mix well so that the concentration of the ruthenium-based photosensitizer in the solution is 20 μM and the concentration of DPBF is 20 μM. Irradiate the solution with a 635 nm laser with a current of 400 mA. Using the ethanol solution of the ruthenium-based photosensitizer with the same concentration as the reference, detect the UV-visible absorption spectrum of DPBF every 5 min. The results are as Figure 4 shown. The absorbance of DPBF gradually decreases with the prolongation of the irradiation time, indicating that this complex can induce singlet oxygen generation under light irradiation.
[0038] Example 4: MTT Assay for Detecting the Effect of the Ruthenium-Based Photosensitizer Prepared in Example 1 on U87-MG Cells
[0039] It is divided into phototoxicity and dark toxicity experiments, and the two experiments are carried out simultaneously.
[0040] Cell grouping: control group, Ru group and Ru+Light group. Seed 4T1 cells at 5000 cells / well in a 96-well plate, place it in a 37 °C constant temperature incubator containing 5% CO 2 and incubate for 24 h, then wash once with PBS. The control group is not treated with other treatments except changing the culture medium. The experimental group changes the culture medium containing different concentrations of the above ruthenium-based photosensitizer. After the Ru+Light group is incubated for 4 h, each well is irradiated (450 nm, 45 J / cm 2 2). The Ru group is not irradiated. After continuing to incubate for 24 h, each well is washed twice with PBS, the culture medium containing 10% MTT is changed, and after incubating for 4 h, the old liquid is discarded. 100 μL of DMSO is added to each well, and the absorbance at 570 nm is detected by machine. Calculate the cell viability through the ratio of the absorbance of the experimental group to the control group. The results are as Figure 5 shown. It can be seen that compared with the Ru group, the Ru+Light group can significantly inhibit cell viability at a lower concentration, indicating that this ruthenium-based photosensitizer shows good photodynamic therapy effect on triple-negative breast cancer cells.
[0041] Example 5: Live / Dead Cell Staining Assay of Triple-Negative Breast Cancer Cells Treated with the Ruthenium-Based Photosensitizer Prepared in Example 1
[0042] Divide 4T1 cells into a control group, a Ru group and a Ru+Light group. Seed them at 50,000 cells / well in a 24-well plate, with 1 well in each group. After seeding, place them in a 37 °C incubator containing 5% CO 2Incubate in a 37 °C constant temperature incubator. After 24 h, replace 0.5 mL of the culture medium in the control group, and replace 0.5 mL of the culture medium containing 1 μM of the sample in the Ru group and the Ru+Light group. Incubate for 4 h after changing the cell culture medium. Irradiate the Ru+Light group with a 450 nm laser (45 J / cm 2 ), and then put it back into the incubator and continue to incubate for 8 h. After incubating for sufficient time, take 1 mL of 1640 basal medium, add 0.5 μL of AM and 0.5 μL of PI, mix well to prepare the staining solution for standby. Aspirate the old solution in the well plate, wash the cells twice with PBS, then add 300 μL of the staining solution to each well, place it in the incubator and incubate in the dark for 20 min. Then aspirate the staining solution, gently wash the cells twice with PBS, add 200 μL of RPMI-1640 basal medium to each well to prevent drying, and take green and red fluorescence images with an EVOS intelligent imaging system. The results are as Figure 6 shown. Compared with other groups, the green fluorescence representing live cells in the Ru+Light group decreased, and the red fluorescence representing dead cells increased, indicating that the ruthenium-based photosensitizer successfully induced the death of triple-negative breast cancer cells under light irradiation.
[0043] Example 6: Apoptosis assay of triple-negative breast cancer cells treated with the ruthenium-based photosensitizer prepared in Example 1
[0044] Divide 4T1 cells into a control group, a Ru group, and a Ru+Light group. Inoculate 100,000 cells per well in a 12-well plate, with 1 well for each group. After seeding, place it in an incubator containing 5% CO 2 at 37 °C. Incubate for 24 h, then wash twice with PBS. Replace the culture medium containing 1 μM of the above ruthenium-based photosensitizer in both groups. Incubate for 4 h after changing the cell culture medium. Irradiate the Ru+Light group with a 450 nm laser (45 J / cm 2 ), and then put it back into the incubator and continue to incubate for 8 h. After incubating for sufficient time, collect the old solution of each group, wash the cells twice with PBS and collect the washing solution. Add 1 mL of trypsin without EDTA to each well, put it back into the incubator and digest for several minutes until most of the cells detach from the wall. Add 2 mL of culture medium to terminate the digestion. Pipette and rinse the bottom of the well, collect all the liquid by group, centrifuge at 2000 rpm for 5 min, discard the supernatant, resuspend with 1 mL of PBS, centrifuge again at 2000 rpm for 5 min, and discard the supernatant for standby. Use a cell apoptosis detection kit. Dilute Binding Buffer (10x) 10-fold in advance for standby. Resuspend each tube of cells with 300 μL of Buffer (1x), add 5 μL of AnnexinV-FITC staining solution, mix well and stain for 15 min. Add 5 μL of PI staining solution and mix well 5 min before loading. Add 200 μL of Buffer (1x) to each tube when loading, and perform relevant operations using CytExpert software. The results are as Figure 7As shown, the apoptosis rate of the Ru+Light group of cells was approximately 74.6%, significantly higher than that of other groups, indicating that this complex can significantly induce apoptosis of triple-negative breast cancer cells under light irradiation. Ru described in the above experiment represents the ruthenium-based photosensitizer prepared in Example 1.
[0045] In summary, the ruthenium-based photosensitizer prepared in the present invention can significantly induce apoptosis of triple-negative breast cancer cells under light irradiation, showing excellent anti-tumor properties, and can be used as a lead drug molecule for anti-tumor treatment, with good development and application prospects in the research and development of anti-tumor drugs.
[0046] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection of the present invention.
Claims
1. A ruthenium-based photosensitizer with light responsiveness, characterized in that: Its cation has the following structure:
2. A ruthenium-based photosensitizer with photoresponsiveness according to claim 1, characterized in that: The cation can further combine with the anion chloride ion to form a ruthenium-based photosensitizer.
3. The method for preparing a ruthenium-based photosensitizer with photoresponsiveness according to claim 1, characterized in that: The following steps are involved: (1) ruthenium trichloride trihydrate, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine and anhydrous lithium chloride are mixed in N,N-dimethylformamide, heated to 150° C. and refluxed for 8 hours. After the reaction is completed, the reaction solution is cooled to room temperature, and pure water is added to the reaction solution until no solid is precipitated. The solid is collected by suction filtration, washed with water, and dried to obtain a crude product of a ruthenium-based photosensitizer; (2) Purifying the sample obtained in step (1) by column chromatography to obtain the ruthenium-based photosensitizer as claimed in claim 1.
4. The method for preparing a ruthenium-based photosensitizer having light responsiveness according to claim 3, characterized in that: In step (1), the molar ratio of ruthenium trichloride trihydrate, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine and anhydrous lithium chloride is 5:15:
23.
5. The method for preparing a ruthenium-based photosensitizer having light responsiveness according to claim 3, characterized in that: In step (2), the adsorbent used in the column chromatography is neutral alumina, and the eluent is a mixture of dichloromethane and ethanol in a volume ratio of 9:
1.
6. Use of a photoresponsive ruthenium-based photosensitizer according to any one of claims 1-2 in photodynamic therapy of triple-negative breast cancer.