A self-reporting photosensitizer, its preparation method and application

Abstract

The application belongs to the technical field of fluorescent probe, and relates to a self-reporting photosensitizer as well as a preparation method and application thereof, and comprises the following steps: 1) under nitrogen protection, IrCl3*3H2O is added into a 2-ethoxyethanol-water solution and mixed; 2-(2,4-difluorophenyl)pyridine is further added, stirred and heated, and then the obtained mixture is subjected to condensation reflux, cooling and vacuum filtration to obtain precipitate; the precipitate is washed and dried to obtain a chloro-bridged dimer; 2) the chloro-bridged dimer is further added into DMSO, and the obtained mixture is subjected to ultrasonic treatment, freeze-drying and separation and purification to obtain the self-reporting photosensitizer. The self-reporting photosensitizer prepared by the application has simple synthesis steps and is easy to operate; the molar absorption coefficient is 6.84*10 4 M ‑1 cm ‑1 , the singlet oxygen yield is 0.45, and the cell phototoxicity index is 15.4; in the application of light-induced cancer cell apoptosis, the fluorescence intensity and fluorescence distribution are observed, and the cell apoptosis is monitored in real time in a double mode, and the living cells and dead cells are obviously distinguished.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, and relates to a self-reporting photosensitizer, its preparation method, and its application. Background Technology

[0002] Cancer is the second leading cause of death worldwide, with 19.29 million new cases reported globally in 2022. Precision medicine has gradually become one of the most attractive research areas in cancer treatment. Currently, photodynamic therapy (PDT) has attracted widespread attention due to its advantages such as non-invasiveness, lack of drug resistance, low toxicity, and high spatiotemporal selectivity. It has already been applied to the treatment of some non-tumor diseases, such as port-wine stains, psoriasis, macular degeneration, and restenosis after angioplasty.

[0003] Photodynamic therapy (PDT) generally refers to the process where, under light irradiation, a photosensitizer converts oxygen into reactive oxygen species (ROS), which then kill cells. However, traditional PDT methods cannot provide real-time feedback on treatment effects, leading to problems such as treatment delays and overtreatment. Imaging-guided PDT, employing a self-reporting photosensitizer system, can monitor the treatment process while generating ROS, thus significantly reducing problems such as treatment delays and overtreatment. Reported self-reporting photosensitizer systems include, for example, "A singletoxygen self-reporting photosensitizer for cancer phototherapy" (Chem. Sci., 2021, 12, 2515); "In vivo therapeutic response monitoring by a self-reporting upconverting covalent organic framework nanoplatform" (Chem. Sci., 2020, 11, 1299); and "A self-reporting AIE probe with a built-in singlet oxygen sensor for targeted photodynamic ablation of cancer cells" (Chem. Sci., 2016, 7, 1862), etc. Although these photosensitizer systems can be used for photodynamic therapy of cancer cells, the following problems exist: (1) The system is complex due to the simultaneous use of photosensitizers and fluorescent dyes; (2) The synthesis steps are complicated, either by covalently bonding photosensitizers with chemical groups sensitive to singlet oxygen to form new self-reporting photosensitizers; or (3) The self-reporting photosensitizer system indicates cell activities such as apoptosis by observing changes in fluorescence intensity, the self-reporting mode is singular and easily affected by factors such as photosensitizer concentration and excitation light intensity. Summary of the Invention

[0004] To address the problems of cumbersome synthesis steps and single mode when indicating cell apoptosis and other activities in existing self-reported photosensitizers, this invention aims to provide a self-reported photosensitizer, its preparation method, and its application. The photosensitizer has a simple and easy synthesis process. When used in photoinduced apoptosis of cancer cells, it can achieve live cell staining of cancer cells and monitor the cell apoptosis process in real time in a dual mode by simultaneously observing changes in fluorescence intensity and cell localization, thus distinguishing between live and dead cells.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing a self-reporting photosensitizer includes the following steps:

[0007] 1) Preparation of chlorinated bridged dimers

[0008] 1.1) Under nitrogen protection, IrCl3·3H2O was added to an aqueous solution of 2-ethoxyethanol and mixed thoroughly; then 2-(2,4-difluorophenyl)pyridine was added, stirred and heated, and then refluxed, cooled and filtered under reduced pressure to obtain a precipitate; the molar ratio of IrCl3·3H2O to 2-(2,4-difluorophenyl)pyridine was 1:2 to 2.2; the volume ratio of IrCl3·3H2O to aqueous solution of 2-ethoxyethanol was 15 mg: 1 mL to 4 mL;

[0009] 1.2) The precipitate was washed and dried to obtain a chlorinated bridged dimer;

[0010] 2) Preparation of self-reporting photosensitizers

[0011] Under nitrogen protection, the chlorinated dimer from step 1) was added to DMSO solvent, with the ratio of chlorinated dimer to DMSO solvent being 30 mg: 5 mL to 6 mL. After sonication, the solvent was removed by freeze-drying. Then, the self-reporting photosensitizer was obtained by chromatography separation and purification.

[0012] Further specifying, the self-reporting photosensitizer in step 2) has the chemical formula C 24 H 18 ClF4IrN2OS, with the following structural formula:

[0013]

[0014] Further specifying, in step 2), the ultrasonic reaction time is 0.5h to 2h; the chromatographic separation is completed in a silica gel column, using dichloromethane and ethanol as developing solvents.

[0015] Further specifying, in step 1.1), the 2-ethoxyethanol-water solution is prepared by mixing 2-ethoxyethanol and water at a volume ratio of 3 to 4:1.

[0016] Further specifying, in step 1.1), the heating temperature is 110℃~120℃; the reaction time is 15h~25h.

[0017] Further specifying, in step 1.2), the precipitate is washed sequentially with deionized water, ethanol, and n-hexane.

[0018] A self-reporting photosensitizer prepared by the aforementioned method.

[0019] The application of self-reporting photosensitizers as fluorescent probes in real-time monitoring of cancer cell apoptosis, as described above.

[0020] Further, by observing the fluorescence intensity and cell localization changes of the self-reported photosensitizer, live cells and dead cells can be clearly distinguished.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] 1. The self-reporting photosensitizer of the present invention is a self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl with 2-(2,4-difluorophenyl)pyridine as the main ligand and DMSO as the auxiliary ligand. The synthesis steps are simple and easy to operate.

[0023] 2. At a wavelength λ = 375 nm, the molar absorptivity of the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl in this invention is relatively large (ε = 6.84 * 10⁻⁶). 4 M -1 cm -1 It has a strong light absorption capacity.

[0024] 3. The self-reporting photosensitizer of the present invention, [(dfppy)2Ir(DMSO)]Cl, has a high singlet oxygen yield (Φ = 0.45, Φ MB =0.52), good biocompatibility (IC50) (黑暗) =118.8 μmol / L), high cytotoxicity (IC50). (光照) =7.7 μmol / L, PI = 15.4). It can generate reactive oxygen species under light irradiation, inducing apoptosis.

[0025] 4. The self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl of this invention can enter HeLa cells. Under light conditions, the fluorescence intensity and intracellular distribution of this self-reporting photosensitizer change with the degree of cell apoptosis. As the degree of apoptosis increases, from early apoptosis to late apoptosis to cell death, the fluorescence intensity gradually increases. The fluorescence distribution changes from the cytoplasm to the nucleus; during cell death, the nucleus emits strong fluorescence. By observing the changes in fluorescence intensity and cell distribution in a dual-mode real-time monitoring of cancer cell apoptosis, the apoptosis process of live cells and dead cells can be clearly distinguished. This dual-mode monitoring system of the self-reporting photosensitizer avoids interference caused by changes in photosensitizer concentration and excitation light intensity, and has potential biological application value. Attached Figure Description

[0026] Figure 1 The mass spectrum of the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl in Example 1;

[0027] Figure 2 The 1H NMR spectrum of the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl in Example 1;

[0028] Figure 3 The crystal structure diagram of the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl in Example 1 is shown.

[0029] Figure 4 The UV-Vis absorption spectrum of the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl in Example 1 is shown below.

[0030] Figure 5 The image shows the electron paramagnetic resonance spectrum of singlet oxygen generated by TEMP capturing photosensitizer under 365nm laser illumination, where (black) represents the unilluminated condition and (red) represents the illuminated condition.

[0031] Figure 6 The graph shows the absorbance of 1,3-diphenylisobenzofuran (DPBF) at 418 nm wavelength as it captures singlet oxygen generated by a photosensitizer under 365 nm laser illumination. (Red) represents DPBF and the photosensitizer [(dfppy)2Ir(DMSO)]Cl; (Black) represents DPBF and the reference compound methylene blue (MB); (Blue) represents DPBF alone.

[0032] Figure 7The graphs show the relationship between the concentration of the photosensitizer [(dfppy)₂Ir(DMSO)]Cl and cell viability. The black graphs show HeLa cells treated with different concentrations (0 μmol / L, 20 μmol / L, 40 μmol / L, 60 μmol / L, 80 μmol / L, 100 μmol / L, 120 μmol / L, and 150 μmol / L) of [(dfppy)₂Ir(DMSO)]Cl for 30 min, followed by incubation in the dark for 24 h. The red graphs show HeLa cells first treated with different concentrations (0 μmol / L, 1.0 μmol / L, 2.0 μmol / L, 4.0 μmol / L, 6.0 μmol / L, 8.0 μmol / L, and 10 μmol / L) of [(dfppy)₂Ir(DMSO)]Cl for 30 min, then exposed to white light (400-700 nm, 25 mW / cm²). 2 Irradiation for 10 minutes, followed by incubation in the dark for 24 hours;

[0033] Figure 8 HeLa cells incubated with the photosensitizer [(dfppy)2Ir(DMSO)]Cl under light (400-700 nm, 25 mW / cm²) 2 Cell imaging images at different times (0 min, 5 min, 10 min and 20 min). Detailed Implementation

[0034] The technical solution of the present invention will be further explained and described below with reference to the accompanying drawings and embodiments, but the present invention is not limited to the embodiments described below.

[0035] The self-reporting photosensitizer of this invention is a self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl with 2-(2,4-difluorophenyl)pyridine as the main ligand and DMSO as the auxiliary ligand, and its chemical formula is C. 24 H 18 ClF4IrN2OS has the following structural formula:

[0036]

[0037] The present invention provides a method for preparing a self-reporting photosensitizer, comprising the following steps.

[0038] 1) Preparation of chlorinated bridged dimers

[0039] 1.1) Under nitrogen protection, IrCl3·3H2O was added to an aqueous solution of 2-ethoxyethanol and mixed thoroughly; then 2-(2,4-difluorophenyl)pyridine was added, stirred and heated, and then refluxed, cooled and filtered under reduced pressure to obtain the precipitate; the molar ratio of IrCl3·3H2O to 2-(2,4-difluorophenyl)pyridine was 1:2 to 2.2; the aqueous solution of 2-ethoxyethanol was prepared by mixing 2-ethoxyethanol and water at a volume ratio of 3 to 4:1; the volume ratio of IrCl3·3H2O to the aqueous solution of 2-ethoxyethanol was 15 mg to 1 mL to 4 mL.

[0040] In this step, the 2-ethoxyethanol-water solution is prepared by mixing 2-ethoxyethanol and water at a volume ratio of 3 to 4:1.

[0041] In this step, the heating temperature is 110℃~120℃; the reaction time is 15h~25h.

[0042] 1.2) The precipitate was washed and dried to obtain chlorobridged dimers; specifically, the precipitate was washed sequentially with deionized water, ethanol and n-hexane.

[0043] 2) Preparation of self-reporting photosensitizers

[0044] Under nitrogen protection, the chlorinated dimer from step 1) was added to DMSO solvent, with the ratio of chlorinated dimer to DMSO solvent being 30 mg: 5 mL to 6 mL. After sonication, the solvent was removed by freeze-drying. Then, the self-reporting photosensitizer was obtained by chromatography separation and purification.

[0045] The ultrasonic reaction time was 0.5 h to 2 h; the chromatographic separation was carried out in a silica gel column, using dichloromethane and ethanol as the developing solvent.

[0046] To further illustrate the performance of the self-reporting photosensitizer prepared by the present invention and its superiority in photodynamic therapy of cancer cells, experimental parameters were randomly selected from the above preparation method for verification experiments. Therefore, the specific embodiments described below should not be used as a limitation on the scope of protection of the present invention.

[0047] It should be noted that the main ligand 2-(2,4-difluorophenyl)pyridine and the auxiliary ligand DMSO used in this invention were purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd.; the HeLa cells used in this invention were all purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; and all products without a specific source are commercially available products.

[0048] Example 1

[0049] This embodiment uses 2-(2,4-difluorophenyl)pyridine as the main ligand and dimethyl sulfoxide (DMSO) as the auxiliary ligand as the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl, and its preparation method includes the following steps.

[0050] 1) Preparation of chlorinated bridged dimers

[0051] Under nitrogen protection, 1.0 mmol of IrCl3·3H2O was added to 40 mL of 75% (v / v) 2-ethoxyethanol aqueous solution and mixed. The mixture was magnetically stirred for 20 min, and then 2.0 mmol of 2-(2,4-difluorophenyl)pyridine was added. The mixture was heated at 110 °C and refluxed for 25 h. After cooling to room temperature, the mixture was filtered under reduced pressure to obtain the precipitate. The precipitate was washed successively with 15 mL of deionized water, 15 mL of ethanol, and 15 mL of n-hexane, and then dried under vacuum to obtain the chlorobridged dimer.

[0052] 2) Preparation of self-reporting photosensitizers

[0053] Under nitrogen protection, 30 mg of the chlorobridged dimer prepared in step 1) was added to 5 mL of DMSO solvent and sonicated at room temperature for 2 h. Then, the solvent was removed by freeze drying. The mixture was then separated by silica gel column chromatography using dichloromethane and ethanol as developing solvents. After purification, a self-reporting photosensitizer with 2-(2,4-difluorophenyl)pyridine as the main ligand and dimethyl sulfoxide (DMSO) as the auxiliary ligand was obtained.

[0054] The chemical formula of the self-reporting photosensitizer in this embodiment, with 2-(2,4-difluorophenyl)pyridine as the main ligand and dimethyl sulfoxide (DMSO) as the auxiliary ligand, is C. 24 H 18 ClF4IrN2OS.

[0055] Mass spectrometry, 1H NMR, and crystal structure analysis were performed on the self-reporting photosensitizer obtained in this embodiment. The results are shown in [reference 1]. Figure 1 , Figure 2 and Figure 3 .

[0056] See Figure 1 The mass spectrum of the self-reported photosensitizer was 651.0697 [M]. + The standard value of the self-reporting photosensitizer, with 2-(2,4-difluorophenyl)pyridine as the main ligand and dimethyl sulfoxide (DMSO) as the auxiliary ligand, is 651.0699 [M]. + near.

[0057] See Figure 2 The self-reported 1H NMR spectrum of the photosensitizer yielded the following results: 1H NMR (600MHz, DMSO-d6) δ9.79 (d, J=5.6Hz, 1H), 9.56 (d, J=5.6Hz, 1H), 8.32 (d, J= 8.5Hz,1H),8.25(d,J=8.4Hz,1H),8.21(t,J=7.6Hz,1H),8.13(t,J=7.7Hz,1H),7 .67(t,J=6.4Hz,1H),7.58(t,J=6.2Hz,1H),6.87(t,J=9.7Hz,1H),6.81(dd,J=1 5.6,5.9Hz,1H),5.72–5.74(m,1H),5.05–5.07(m,1H),3.15(s,3H),2.09(s,3H).

[0058] See Figure 3 The crystal structure diagram of the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl is shown. This crystal structure indicates that the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl forms an orthorhombic crystal, with the iridium atom also exhibiting distorted octahedral coordination, coordinated to one S atom and one Cl atom of DMSO.

[0059] Example 2

[0060] The preparation method of the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl with 2-(2,4-difluorophenyl)pyridine as the main ligand and dimethyl sulfoxide (DMSO) as the auxiliary ligand in this embodiment includes the following steps.

[0061] 1) Preparation of chlorinated bridged dimers

[0062] Under nitrogen protection, 1.0 mmol of IrCl3·3H2O was added to 30 mL of 80% (v / v) 2-ethoxyethanol aqueous solution and mixed. The mixture was magnetically stirred for 20 min. Then, 2.1 mmol of 2-(2,4-difluorophenyl)pyridine was added, and the mixture was heated at 115 °C and refluxed. The mixture was stirred for 20 h and then cooled to room temperature and filtered under reduced pressure to obtain the precipitate. The precipitate was washed successively with 15 mL of deionized water, 15 mL of ethanol, and 15 mL of n-hexane, and dried under vacuum to obtain the chlorobridged dimer.

[0063] 2) Preparation of self-reporting photosensitizers

[0064] Under nitrogen protection, 30 mg of the chlorobridged dimer prepared in step 1) was added to 5.5 mL of DMSO solvent and sonicated at room temperature for 1 h. Then, the solvent was removed by freeze drying. The mixture was then separated by silica gel column chromatography using dichloromethane and ethanol as developing solvents. After purification, a self-reporting photosensitizer with 2-(2,4-difluorophenyl)pyridine as the main ligand and dimethyl sulfoxide (DMSO) as the auxiliary ligand was obtained.

[0065] Example 3

[0066] This embodiment uses 2-(2,4-difluorophenyl)pyridine as the main ligand and dimethyl sulfoxide (DMSO) as the auxiliary ligand as the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl, and its preparation method includes the following steps.

[0067] 1) Preparation of chlorinated bridged dimers

[0068] Under nitrogen protection, 1.0 mmol of IrCl3·3H2O was added to 50 mL of 80% 2-ethoxyethanol aqueous solution and mixed. The mixture was magnetically stirred for 15 min. Then, 2.2 mmol of 2-(2,4-difluorophenyl)pyridine was added and the mixture was heated at 120 °C and refluxed. The mixture was stirred for 15 h and then cooled to room temperature and filtered under reduced pressure to obtain the precipitate. The precipitate was washed sequentially with 15 mL of deionized water, 15 mL of ethanol and 15 mL of n-hexane, and dried under vacuum to obtain the chlorobridged dimer.

[0069] 2) Preparation of self-reporting photosensitizers

[0070] Under nitrogen protection, 30 mg of the chlorobridged dimer prepared in step 1) was added to 6 mL of DMSO solvent and sonicated at room temperature for 0.5 h. Then, the solvent was removed by freeze drying. The mixture was then separated by silica gel column chromatography using dichloromethane and ethanol as developing solvents. After purification, a self-reporting photosensitizer with 2-(2,4-difluorophenyl)pyridine as the main ligand and dimethyl sulfoxide (DMSO) as the auxiliary ligand was obtained.

[0071] The performance of the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl, prepared in the above examples with 2-(2,4-difluorophenyl)pyridine as the main ligand and DMSO as the auxiliary ligand, is verified below.

[0072] Verification Experiment 1

[0073] Taking the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl prepared in Example 1 as an example, the molar absorptivity of the self-reporting photosensitizer was verified.

[0074] The experimental procedure was as follows: A solution with pH = 7.4 was prepared by mixing 10 mmol / L phosphate buffer (8.2 mmol / L NaH2PO4, 1.8 mmol / L Na2HPO4, PB) and dimethyl sulfoxide at a volume ratio of 99:1. The UV-Vis absorption spectrum of the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl was measured, and the results are as follows. Figure 4 As shown.

[0075] See Figure 4 The self-reporting photosensitizer [(dfppy)₂Ir(DMSO)]Cl exhibits strong absorption in the wavelength range below 330 nm and weak absorption in the wavelength range of 330–500 nm, which is due to charge transfer from the metal to the ligand. Using an absorption wavelength of 375 nm, the molar absorptivity of the self-reporting photosensitizer [(dfppy)₂Ir(DMSO)]Cl in the buffer system at this wavelength was calculated to be ε = 6.84 * 10⁻⁶. 4 M -1 cm -1 It can be seen that the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl prepared in this invention has a large molar absorptivity.

[0076] Verification Experiment 2

[0077] Taking the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl prepared in Example 1 as an example, its singlet oxygen yield was verified.

[0078] The experimental procedure was as follows: An acetonitrile solution containing 10 μmol / L of the self-reporting photosensitizer [(dfppy)₂Ir(DMSO)]Cl and 20 mmol / L of TEMP (2,2,6,6-tetramethylpiperidine) was placed in a capillary tube (Φ = 1 mm, h = 5 cm). One end was then sealed with Vaseline and inserted into the tube along the edge of the paramagnetic tube. The electron paramagnetic resonance spectrum of singlet oxygen generated by the photosensitizer was captured under 365 nm laser illumination. The results are as follows: Figure 5 As shown. (Black) represents the condition without light; (red) represents the condition with light.

[0079] See Figure 5 White light irradiation (400-700nm, 20mW / cm) 2 It can be clearly seen after 10 minutes. 1 The triplet signal induced by O2 was observed, while no obvious signal was observed without white light irradiation, indicating that [(dfppy)2Ir(DMSO)]Cl can indeed be used as a photosensitizer.

[0080] A DMSO solution containing 10 μmol / L of the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl and 50 μmol / L of 1,3-diphenylisobenzofuran (DPBF) was irradiated at 365 nm for different times, and the absorbance at 418 nm was recorded. The absorbance changes were plotted against the irradiation time to obtain the absorbance change at 418 nm for (DPBF) capturing singlet oxygen generated by the photosensitizer under 365 nm laser irradiation. The results are as follows: Figure 6As shown. (Red) represents DPBF and the self-reporting photosensitizer; (Black) represents DPBF and the reference compound methylene blue (MB); (Blue) represents DPBF alone.

[0081] The absorbance of the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl and methylene blue at 365 nm was adjusted to approximately 0.05. The Φ of the self-reporting photosensitizer... Δ Calculate using the following formula.

[0082]

[0083] Where the subscripts x and std represent the self-reporting photosensitizer and methylene blue, respectively, S is the slope of the absorbance change of DPBF at a wavelength of 418 nm over time, and F is the absorption correction factor, F = 1-10. -OD (OD is the optical density of the self-reporting photosensitizer and methylene blue at 365 nm).

[0084] from Figure 6 As can be seen, with the increase of illumination time, the mixture containing both the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl and DPBF exhibits the largest slope in the change of absorbance at 418 nm over time, and its singlet oxygen yield is as high as 0.45 (Φ MB =0.52), which has a high singlet oxygen yield.

[0085] Verification Test 3: Cytotoxicity Verification

[0086] Solutions containing the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl prepared in Example 1 were prepared at concentrations of 0 μmol / L, 1 μmol / L, 2 μmol / L, 4 μmol / L, 6 μmol / L, 8 μmol / L, 10 μmol / L, 20 μmol / L, 40 μmol / L, 60 μmol / L, 80 μmol / L, 100 μmol / L, 120 μmol / L, and 150 μmol / L, respectively.

[0087] 100 μL, 5 × 10 4Cells in the logarithmic growth phase (cells / mL) were added to the wells of a 96-well plate and cultured for 24 hours. HeLa cells were then divided into two groups: one group was incubated for 0.5 hours in cell culture medium containing different concentrations of self-reported photosensitizers (0-150 μmol / L) to detect dark cytotoxicity; the other group was incubated for 0.5 hours in cell culture medium containing different concentrations of self-reported photosensitizers (0-10 μmol / L) followed by 10 min of white light irradiation to detect phototoxicity. After removing the culture medium containing different concentrations of self-reported photosensitizers, the cells were washed twice with PBS, and then 10 μL of CCK-8 solution was added to each well. The cells were incubated at 37°C and 5% CO2 for 4 hours. The absorbance (A) of the liquid in each well of the 96-well plate at 450 nm was recorded using a microplate reader. 样品处理组 Wells containing cells, the appropriate amount of cell culture medium, and CCK-8 solution but without the drug served as a blank control group, and the absorbance at 450 nm was recorded according to the above steps (A). 空白对照组 According to the formula

[0088]

[0089] To obtain cell activity under different conditions, such as Figure 7 As shown.

[0090] See Figure 7 After incubating HeLa cells with 60 μmol / L of the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl for 24 h, the cell viability was >80%. The IC50 of the self-reporting photosensitizer on HeLa cells under dark conditions was calculated using Graphpad Prism software. 50 The value was 118.8 μM, indicating that the self-reporting photosensitizer had good cell compatibility. Another group of HeLa cells were exposed to white light (20 mW / cm²) for 10 min. 2 Cells were treated with CCK-8 and then the IC50 under illumination was calculated using Graphpad Prism software. 50 The value was 7.7 μM, indicating that the self-reporting photosensitizer has high phototoxicity.

[0091] Therefore, the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl prepared in this invention, with 2-(2,4-difluorophenyl)pyridine as the main ligand and dimethyl sulfoxide (DMSO) as the auxiliary ligand, exhibits high biocompatibility when incubated with HeLa cells, resulting in minimal damage to the cells. However, when the cells are exposed to light, they suffer severe damage. This indicates that the fluorescent probe is a relatively ideal photosensitizer.

[0092] The self-reporting photosensitizers [(dfppy)2Ir(DMSO)]Cl, prepared in Examples 2 and 3 with 2-(2,4-difluorophenyl)pyridine as the main ligand and DMSO as the auxiliary ligand, were tested for molar absorptivity, singlet oxygen yield, and cytotoxicity. The results were the same as those in Example 1, indicating that the photosensitizers prepared by the method of the present invention have high molar absorptivity, high singlet oxygen yield, low dark cytotoxicity, and high phototoxicity.

[0093] Verification Experiment 4

[0094] The apoptosis process of cancer cells during photodynamic therapy was monitored using the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl prepared in Example 1, with 2-(2,4-difluorophenyl)pyridine as the main ligand and dimethyl sulfoxide (DMSO) as the auxiliary ligand. The specific operation steps are as follows.

[0095] First, digested HeLa cells are divided into groups of 1×10⁻⁶. 5 HeLa cells were seeded at 10 mW / mL in glass-bottomed culture dishes and cultured in 1 mL of fresh cell culture medium for 24 h. After removing excess medium, the cells were incubated with cell culture medium containing 10 μmol / L of the self-reporter photosensitizer [(dfppy)₂Ir(DMSO)]Cl at 37 °C for 30 min. The cells were then washed three times with 10 mmol / L PBS and cultured in 1 mL of fresh cell culture medium. The obtained HeLa cells were then subjected to a 20 mW / cm² hydrochloric acid treatment. 2 White light irradiation was performed for 0 min, 5 min, 10 min, and 20 min, respectively. After treatment, the cells were incubated with 2 μmol / L Annexin V-FITC and 5 μmol / L PI for 30 min, respectively. Subsequently, the HeLa cells were washed three times with 10 mmol / L PBS and placed in 1 mL of fresh cell culture medium without phenol red for laser scanning confocal microscopy imaging. The excitation wavelength was set to 405 nm, and the acquisition wavelength range was 430 nm–530 nm. Fluorescence imaging images were recorded and analyzed, and the results are as follows: Figure 8 As shown.

[0096] See Figure 8During imaging, after incubating HeLa cells with the self-reported photosensitizer [(dfppy)2Ir(DMSO)]Cl for 30 min without light exposure, very weak blue fluorescence emission was observed in the HeLa cells (b), with no green (i) or red (m) fluorescence emission, indicating that the HeLa cells were alive in the presence of the self-reported photosensitizer. After 5 min of light exposure, weak blue fluorescence emission from the self-reported photosensitizer channel (f) was observed, and weak green fluorescence emission appeared in some cell membranes (j), with membrane bubbling, and the PI signal was still not visible (n), indicating that the cells were in the early stage of apoptosis. After 10 min of light exposure, almost all cells showed green (k) fluorescence emission, and some cells showed red (o) fluorescence emission, indicating that most cells had died. More notably, the self-reported photosensitizer channel showed obvious blue fluorescence emission, and in the overlay image, the blue emitting cell area basically overlapped with the bright-field cytoplasm (s), indicating that the self-reported photosensitizer can directly detect dead cells. After 20 minutes of light irradiation, the entire cell exhibited blue fluorescence emission, while all cells showed both green (l) and red (p) fluorescence emission. Notably, after 10 minutes of light irradiation, the blue fluorescence emission was observed in the cytoplasm (s), and after 20 minutes, it appeared throughout the entire cell (t). This demonstrates that the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl, with 2-(2,4-difluorophenyl)pyridine as the main ligand and dimethyl sulfoxide (DMSO) as the auxiliary ligand, can not only act as a photosensitizer to induce apoptosis by generating reactive oxygen species within the cell under light irradiation, but also serve as a fluorescent probe to self-report the apoptosis process and distinguish between live and dead cells, thus possessing potential biological application value.

[0097] Verification has shown that the self-reporting photosensitizer [(dfppy)2Ir(DMSO)]Cl, with 2-(2,4-difluorophenyl)pyridine as the main ligand and dimethyl sulfoxide (DMSO) as the auxiliary ligand, prepared in this invention, possesses a large molar absorptivity, high singlet oxygen yield, and low biotoxicity. It can be used as a self-reporting photosensitizer to induce reactive oxygen species in cells under light irradiation, thereby inducing apoptosis in cancer cells. Furthermore, it can serve as a fluorescent probe to report the apoptosis process of cells, distinguishing between live and dead cells, and enabling dual-mode monitoring of cancer cell apoptosis during photodynamic therapy, demonstrating potential biological application value.

Claims

1. A method of preparing a self-reporting photosensitizer, comprising the steps of: 1) preparing a chloro-bridged dimer 1.1) under the protection of nitrogen, IrCl3·3H2O is added into 2-ethoxyethanol-water solution and mixed uniformly; then 2-(2,4-difluorophenyl)pyridine is added, stirred and heated, and then condensed and refluxed, cooled and filtered under reduced pressure to obtain precipitate; the molar ratio of IrCl3·3H2O to 2-(2,4-difluorophenyl)pyridine is 1:2-2.2; the ratio of the amount of IrCl3·3H2O to 2-ethoxyethanol-water solution is 15mg:1mL-4mL; 1.2) the precipitate is washed and dried to obtain chloro-bridged dimer; 2) preparation of self-reporting photosensitizer under the protection of nitrogen, the chloro-bridged dimer of step 1) is added into DMSO solvent, the ratio of the amount of chloro-bridged dimer to DMSO solvent is 30mg:5mL-6mL; after ultrasonic treatment, the solvent is removed by freeze-drying; then chromatographic separation and purification are carried out to obtain self-reporting photosensitizer; The self-reporting photosensitizer has a chemical formula of C 24 H 18 ClF4IrN2OS, and a structural formula is:

2. The method for preparing the self-reporting photosensitizer according to claim 1, characterized in that, in step 2), the ultrasonic reaction time is 0.5h-2h; the chromatographic separation is carried out in a silica gel column, and dichloromethane and ethanol are used as developing agents.

3. The method for preparing the self-reporting photosensitizer according to claim 1, characterized in that, in step 1.1), the 2-ethoxyethanol-water solution is prepared by mixing 2-ethoxyethanol and water in a volume ratio of 3-4:

1.

4. The method for preparing the self-reporting photosensitizer according to claim 1, characterized in that, in step 1.1), the heating temperature is 110℃-120℃; the reaction time is 15h-25h.

5. The method for preparing the self-reporting photosensitizer according to claim 1, characterized in that, in step 1.2), the precipitate is washed with deionized water, ethanol and n-hexane in sequence.

6. The self-reporting photosensitizer prepared by the preparation method of the self-reporting photosensitizer according to claim 1.

7. The self-reporting photosensitizer according to claim 6 as a fluorescent probe for preparing a product for real-time monitoring of cancer cell apoptosis.

8. Use of a self-reporting photosensitizing agent according to claim 7, characterized in that, When the self-reporting photosensitizer is used in the preparation of a product for real-time monitoring of cancer cell apoptosis, the luminescent intensity and cell localization change of the self-reporting photosensitizer fluorescence are observed to distinguish live cells and dead cells obviously.

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