Non-oxygen-dependent multimodal selenium-rhodamine-based photodynamic photosensitizers, preparation and use thereof

Abstract

The application belongs to the technical field of fluorescent probe, and particularly relates to a non-oxygen-dependent multi-mode selenium rhodamine-based photodynamic photosensitizer as well as preparation and application. In order to develop a non-oxygen-dependent and efficient photosensitizer, based on the selenium rhodamine spiro ring 'on-off' mechanism, a N-nitroso functionalized selenium rhodamine photosensitizer SeR-NO is developed, which exists in the form of lactone with closed ring in PBS, has very small cell dark toxicity and excellent cell permeability; under light, the photosensitizer can not only produce a large amount of ROS through type I and type II mechanism, but also can catalyze NADPH oxidation and Cyt c reduction in an oxygen-independent manner, therefore, SeR-NO has large phototoxicity, and can induce cell death through ferroptosis in normoxia (21% O2) and hypoxia (2% O2).

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to a non-oxygen-dependent multimode selenodamine-based photodynamic photosensitizer and its preparation and application. Background Technology

[0002] Photodynamic therapy (PDT) is a photochemically regulated treatment method that utilizes the combined action of photosensitizers (PSs), light, and oxygen (O2) to generate reactive oxygen species (ROS), thereby achieving therapeutic effects. Specifically, under light irradiation, PSs are excited from the ground state (S0) to the excited singlet state (S1), and then decay to the excited triplet state (T1) via intersystem crossing (ISC). Due to its long lifetime, the generated T1-state photosensitizer can directly transfer its energy to the surrounding O2 via triplet-triplet energy transfer, generating highly cytotoxic singlet oxygen (ROS). 1 O2 (Type II mechanism); or it may undergo a cascade electron reaction or hydrogen extraction reaction with adjacent biomolecules to generate superoxide (O2). ·- Active intermediates such as hydroxyl radicals (OH·) and hydroxyl radicals (Type I mechanism) induce apoptosis and / or necrosis of cancer cells.

[0003] Most reported or clinically used photosensitizers are type II photosensitizers, which are highly dependent on oxygen concentration. However, due to the rapid proliferation of tumor cells, the tumor interior is in an extremely hypoxic environment (pO2 < 5 mmHg), thus greatly limiting the application of type II photosensitizers in solid tumors. In contrast, type I photosensitizers have been shown to be less dependent on oxygen. Recent studies have shown that some photosensitizers (PSs) can photocatalyze the oxidation of NADPH, thereby altering the intracellular NADH / NAD ratio. + The balance ultimately leads to cell death, a strategy that is oxygen-independent. Furthermore, the combined use of photothermal therapy (PDT) with other treatment modalities (such as photothermal therapy, immunotherapy, and gas therapy) can significantly improve tumor treatment efficacy and overcome many shortcomings of single PDT (such as tumor resistance, proliferation, metastasis, and high oxygen dependence), providing new insights for developing oxygen-independent, highly efficient photosensitizers. Summary of the Invention

[0004] Selenodazole and its derivatives are structurally natural photodynamic photosensitizers, primarily due to the ISC process promoted by the selenium atom in their molecular structure. However, as members of the rhodamine dye family, photodynamic photosensitizers developed based on the 2-carboxyphenyl selenodazole spirocyclic "close-open" mechanism are rarely reported. Based on this, this invention develops an N-nitrosofunctionalized selenodazole photosensitizer, SeR-NO. This photosensitizer exists in PBS as a cyclic-closed lactone, exhibiting minimal dark cytotoxicity and excellent cell permeability. Under light irradiation, it not only generates large amounts of ROS through type I and type II mechanisms but also catalyzes NADPH oxidation and Cyt c reduction in an oxygen-independent manner. Therefore, SeR-NO exhibits high phototoxicity, inducing cell death via ferroptosis under both normoxic (21% O2) and hypoxic (2% O2) conditions.

[0005] To achieve the above objectives, the present invention employs the following technical solution:

[0006] This invention provides a non-oxygen-dependent, multi-mode selenorodamine-based photodynamic photosensitizer, with the following structural formula:

[0007]

[0008] This invention also provides a method for preparing a non-oxygen-dependent multimode selenorodamine-based photodynamic photosensitizer, comprising the following steps:

[0009]

[0010] Step 1: Under nitrogen protection, 2-iodobenzoic acid and anhydrous THF were mixed and the resulting solution was cooled. Then, isopropyl magnesium chloride-lithium chloride solution was added dropwise to react. After heating, selenopyrrolidone THF solution was added dropwise to continue the reaction. The mixture was gradually restored to room temperature to continue the reaction. After the reaction was completed, the reaction solution was quenched under ice-water bath conditions, the solvent was evaporated, and the resulting solid was redissolved in a mixed solution of concentrated hydrochloric acid and methanol and the reaction was continued with stirring. The solvent was removed by vacuum distillation of the mixture, and the crude product was separated by chromatographic column to obtain 2-carboxyphenylselenopyrrolidone.

[0011] Step 2: Dissolve 2-carboxyphenylselenodazole in dichloromethane, then add sodium hydroxide solution. The mixture is extracted, dried, filtered, and evaporated under reduced pressure to obtain a yellow oil. Dissolve the yellow oil in ultra-dry THF, add n-butyl nitrite, and stir to react. After the reaction is complete, evaporate the solvent under reduced pressure. The crude product is purified by silica gel column chromatography to obtain the photosensitizer.

[0012] Furthermore, in step 1, the molar ratio of 2-iodobenzoic acid, isopropyl magnesium chloride-lithium chloride, and selenopyronone is 10:20:1.

[0013] Furthermore, in step 1, the temperature is cooled to -78°C, and the isopropyl magnesium chloride-lithium chloride solution is added dropwise for 30 minutes. After the temperature is raised to 0°C, the THF solution of selenopyronin is added dropwise for another 30 minutes. The temperature is then gradually restored to room temperature and the reaction continues for 48 hours.

[0014] Furthermore, the feature is that the volume ratio of concentrated hydrochloric acid to methanol in step 1 is 1:11.

[0015] Furthermore, the feature is that the developing solvent used in step 1 is DCM and MeOH, with a volume ratio of 20:1.

[0016] Furthermore, the characteristic feature is that the molar ratio of 2-carboxyphenylselenomethoxyrhodamine and n-butyl nitrite in step 2 is 1:3.

[0017] Furthermore, in step 2, the sodium hydroxide solution has a mass fraction of 5%, the stirring temperature is 60°C, and the reaction time is overnight.

[0018] Furthermore, in step 2, the developing solvent used for silica gel column chromatography is PE and EA, with a volume ratio of 10:1.

[0019] This invention also provides an application of a non-oxygen-dependent multimode selenorodamine-based photodynamic photosensitizer to kill cancer cells under 561nm LED light source irradiation.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] This invention develops a photodynamic photosensitizer SeR-NO based on the spirocyclic "close-on" mechanism of selenopyridine. Specifically, 2-iodobenzoic acid is reacted with isopropyl magnesium chloride-lithium chloride solution, and the resulting Grignard reagent undergoes a nucleophilic addition / elimination reaction with selenopyrone to obtain 2-carboxyphenylselenopyridine in 69.6% yield. 2-Carboxyphenylselenopyridine is then reacted with n-butyl nitrite to finally obtain the photosensitizer SeR-NO in 77% yield.

[0022] Under 565nm LED light irradiation, SeR-NO rapidly releases NO· and the photosensitizer SeR. The released SeR simultaneously generates a large amount of O2 through both type I and type II mechanisms. ·- and 1 O2, O2 ·- SeR-NO can further undergo a cascade reaction with NO· to ​​generate the more toxic ONOO-. Importantly, SeR-NO can also catalyze the oxidation of NADPH and the reduction of Cyt c in an oxygen-independent manner under light irradiation.

[0023] CCK8 experiments showed that SeR-NO can induce A549 cell death via ferroptosis, with IC50 values ​​of 1.02 μM and 0.97 μM under normoxic (21% O2) and hypoxic (2% O2) conditions, respectively. Attached Figure Description

[0024] Figure 1 SeR-NO (2 μM) was prepared in PBS using LED light sources of different wavelengths (10 mW / cm²). 2 Changes in fluorescence spectrum under irradiation.

[0025] Figure 2 Comparison of HPLC-MS chromatograms before and after SeR-NO (5μM) irradiation.

[0026] Figure 3 The amount of NO· released by SeR-NO (2 μM) after light irradiation was determined using Griess reagent.

[0027] Figure 4 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA) under laser (565 nm, 10 mW / cm) 2 The UV-Vis absorption spectrum changes of singlet oxygen generated by capturing photosensitizers under irradiation, where (A) represents ABDA and SeR-NO, and (B) represents ABDA and RB.

[0028] Figure 5 (A) represents DHR 123 in a laser (565nm, 10mW / cm²). 2 (A) Fluorescence spectrum changes of O71 under laser irradiation (565nm, 10mW / cm²) to capture photosensitizer and generate superoxide; (B) Fluorescence spectrum changes of O71 under laser irradiation (565nm, 10mW / cm²) to capture photosensitizer and generate superoxide. 2 Fluorescence spectrum changes of peroxynitrite anions generated by capturing photosensitizers under irradiation.

[0029] Figure 6 In Figures (A) and (B), the images show the capture of TEMP (in PBS) and DMPO (in methanol), respectively. 1 O2 and O2 ·- EPR spectrum of / NO·.

[0030] Figure 7 In the diagram, (A) and (B) represent SeR-NO produced in oxygen-saturated PBS and nitrogen-saturated PBS, respectively. 1 O2、、O2 ·- and ONOO - The situation.

[0031] Figure 8In the image, (A) and (B) are oxygen-saturated PBS solutions with SeR-NO (15 μM) / NADPH (240 μM) and nitrogen-saturated PBS, respectively, under illumination (565 nm LED, 10 mW / cm²). 2 (c) and (d) are the UV-Vis absorption spectra of PBS solutions saturated with oxygen (SeR-NO (15 μM) / NADPH (240 μM) / Cyt c (60 μM)) and nitrogen (10 mW / cm²) respectively, under illumination (565 nm LED, 10 mW / cm²). 2 The graph shows the changes in the ultraviolet-visible absorption spectrum.

[0032] Figure 9 (A) shows confocal images of SeR-NO-loaded A549 cells at different time points under continuous irradiation with a 561nm laser; (B) shows a confocal comparison of SeR-NO-loaded A549 cells after non-illumination and illumination (20 minutes). Collection range: 570-700nm (λex=561nm), scale bar: 20μm.

[0033] Figure 10 Confocal images of cells loaded with DCFH-DA / SeR-NO, MCR-DMA / SeR-NO, DHE / SeR-NO, DAF-FM DA / SeR-NO, or O71 / SeR-NO, after illumination or without illumination. The collection ranges are: 500-550 nm (λex = 488 nm), 650-750 nm (λex = 633 nm), 570-630 nm (λex = 561 nm), 500-540 nm (λex = 488 nm), and 500-550 nm (λex = 488 nm), respectively. Scale bar: 20 μm.

[0034] Figure 11 The phototoxicity and dark toxicity experiments of SeR-NO were conducted under normoxic and hypoxic conditions, respectively.

[0035] Figure 12 Cell viability graph of A549 cells loaded with SeR-NO and different cell death inhibitors.

[0036] Figure 13 The image shows the results of live / dead cell staining experiments on A549 cells treated with SeR-NO, SeR-NO+hν, and SeR-NO+Fer-1+hν. The collection ranges for Calcein-AM and PI are 490-560 nm (λex = 488 nm) and 570-750 nm (λex = 561 nm), respectively. Scale bar: 20 μm. Detailed Implementation

[0037] To further illustrate the technical solution of the present invention, the present invention will be further described below through embodiments.

[0038] Example 1

[0039] A non-oxygen-dependent, multimodal selenorhodamine-based photodynamic photosensitizer, with the following structural formula:

[0040]

[0041] A method for preparing a non-oxygen-dependent, multimodal selenorhodamine-based photodynamic photosensitizer includes the following steps:

[0042] Step 1: 2-Iodobenzoic acid (719.2 mg, 2.9 mmol) was added to a dry round-bottom flask. Anhydrous THF (2.0 mL) was added under nitrogen protection, and the resulting solution was cooled to -78°C. Then, isopropyl magnesium chloride-lithium chloride solution (1.3 M, 4.5 mL, 5.8 mmol) was added dropwise. The reaction solution was reacted at -78°C for 30 minutes, then heated to 0°C. A THF solution of selenopyroninone (100 mg, 0.29 mmol) was then added dropwise. The reaction solution was reacted at 0°C for 30 minutes, then gradually restored to room temperature. The reaction was carried out at room temperature for 48 hours. After the reaction was complete, the reaction solution was quenched with methanol (5.0 mL) in an ice-water bath. After the solvent was evaporated, the solid was redissolved in a mixed solution of concentrated hydrochloric acid and methanol (5.0 mL, volume ratio 1:11), and stirring was continued for 10 minutes. The solvent was removed from the mixture by vacuum distillation, and the crude product was separated by chromatographic column (DCM / MeOH = 20 / 1, V / V) to obtain 2-carboxyphenylselenodazole, which was a blue-purple solid (98.0 mg, 69.6%).

[0043] 1 H NMR(600Hz, CDCl3)7.95(d,J=7.8Hz,1H),7.83(d,J=7.8Hz,1H),7.62(t,J=7.8Hz,1H),7.54(t,J=7.2H z,1H),7.02(d,J=9.0Hz,2H),6.91(d,J=2.4Hz,2H),7.02(dd,J1=8.4Hz,J2=2.4Hz,2H),2.96(s,12H); 13 C NMR (150MHz, CDCl3) δ170.4,150.1,134.1,130.2,130.1,129.3,127.8,125.8,125.6,124.1,122.4,111.6,110.9,40.2; ESI-MS:[M+H] + calcd for451.09193,Found 451.09107.

[0044] Step 2: 2-Carboxyphenylselenosylrhodamine (100.0 mg, 0.2 mmol) was dissolved in DCM (10.0 mL), followed by the addition of 5% sodium hydroxide (NaOH) solution (20.0 mL). The mixture was extracted three times with DCM. The organic phase was dried over anhydrous Na₂SO₄, filtered, and evaporated under reduced pressure to obtain a yellow oil. The yellow oil was dissolved in ultradry THF (15.0 mL), and n-butyl nitrite (n-BuONO) (64.9 mg, 0.6 mmol) was added. The mixture was stirred overnight at 60 °C. After the reaction was complete, the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (PE / EA = 10 / 1, V / V) to obtain SeR-NO, a white solid (74.7 mg, 77%).

[0045] 1 H NMR (600MHz, DMSO-d6) δ8.10(d,J=2.3Hz,1H),7.99(d,J=7.6Hz,1H),7.85(dt,J=14.2,7.4Hz,2H),7.72(t,J=7.3Hz,1H),7.54(dd, J=8.7,2.3Hz,1H),7.21(s,1H),7.08(d,J=2.5Hz,1H),6.90(d,J=9.0Hz,1H),6.61(dd,J=9.0,2.6Hz,1H),3.42(s,3H),2.92(s,6H); 13 C NMR(150MHz,DMSO-d6)δ169.53,153.02,150.64,142.23,135.82,133.41,130.91,130.73,129.35,1 28.01,126.26,124.65,124.42,120.32,119.90,118.07,111.84,111.71,88.33,31.61; HR-MS[M+H] + :calculated for 466.0664,Found 466.0666.

[0046] Example 2

[0047] 1. Solution preparation

[0048] A stock solution (2 mM) of SeR-NO was prepared using chromatographically pure CH3CN and diluted to the required concentration with PBS (10 mM, pH 7.4) before testing. 9,10-Anthracenediyl-di(methylene)dimalonic acid (ABDA, 5 mM), NADPH (50 mM), and Cytc (5 mM) were prepared using ultrapure water. Solutions of DHR123 (2 mM) and DHE (2 mM) were prepared using chromatographically pure DMF. Cell death inhibitors (10 mM) were prepared using sterile DMSO, including necrosis inhibitor (Necrostatin-1, Nec-1), ferroptosis inhibitor (Fer-1), apoptosis inhibitor (Z-VAD-FMK), and autophagy inhibitor 3-methyladenine (3-MA).

[0049] 2. In vitro performance study of photosensitizers

[0050] First, the photoactivation ability of SeR-NO (2 μM) in PBS was investigated using a fluorescence spectrophotometer. Figure 1 As shown, SeR-NO exists in PBS in the form of a cyclic lactone, when used at wavelengths of 375 nm, 450 nm, 505 nm, 565 nm, and 625 nm (10 mW / cm²). 2 After irradiation of SeR-NO with an LED light source, the fluorescence at 595 nm was significantly enhanced, and the fluorescence intensity reached equilibrium in about 30 seconds, with the solution color changing from colorless to purple. HPLC-MS results confirmed that SeR-NO, after light irradiation, generated the NO-depleted product SeR( Figure 2 Next, Griess reagent was used to verify that SeR-NO (2 μM) could release NO·(2 μM) with an efficiency of approximately 100% under light irradiation. Figure 3 ).

[0051] Since the maximum absorption wavelength of the photosensitizer SeR is approximately 565 nm, a 565 nm LED light source was chosen as the final excitation source. In other words, under 565 nm LED light irradiation, SeR-NO can rapidly release NO· and the photosensitizer SeR. The released SeR immediately generates highly reactive oxides (ROS) under illumination, thus achieving the simultaneous spatial and temporal release of NO· and other ROS.

[0052] 3. Study on the ability of photosensitizers to generate reactive oxygen species under normoxic / hypoxic conditions

[0053] First, using ABDA as 1 O2 scavengers, using rose red (RB) as a reference, were used to test the production of SeR-NO in PBS. 1 The ability of O2. For example... Figure 4 As shown in (A), a 565nm LED light source (10mW / cm²) was used. 2When ABDA / SeR-NO solution (or ABDA / RB solution) was continuously irradiated with light at 10-second intervals, the absorption peak at 565 nm gradually increased, while the absorbance of ABDA at 380 nm rapidly decreased. The experimental results indicate that SeR-NO releases the photosensitizer SeR under illumination and simultaneously generates a large amount of... 1 O2, singlet oxygen production efficiency is significantly stronger than RB ( Figure 4 (B)

[0054] Subsequently, the commercial fluorescent probe DHR 123 was used to test the O2 production of SeR-NO in PBS. ·- The ability. For example... Figure 5 As shown in (A), when using a 565nm LED light source (565nm LED, 10mW / cm²), 2 When DHR 123 / SeR-NO solution was continuously irradiated (with 10-second intervals), the fluorescence intensity at 530 nm gradually increased, indicating that SeR-NO can generate a large amount of O2 under illumination. ·- It is worth noting that SeR-NO releases O2 in both space and space under illumination. ·- SeR-NO can undergo a cascade reaction with NO· to ​​generate the more toxic ONOO-. Next, the production of ONOO- by SeR-NO under illumination was tested using the commercially available fluorescent probe O71. - The ability. For example... Figure 5 As shown in (B), when using a 565nm LED light source (565nm LED, 10mW / cm²), 2 When O71 / SeR-NO solution was continuously irradiated at 10-second intervals, the fluorescence at 530 nm was significantly enhanced, confirming the presence of ONOO. - The above experimental results confirm that SeR-NO can generate large amounts of [products] through both Type I and Type II pathways. 1 O2 and O2 ·- O2 ·- It can further react with NO· released in space and time to generate more toxic ONOO-.

[0055] Furthermore, the free radicals generated by SeR-NO under illumination were identified using EPR spectroscopy. First, 2,2,6,6-tetramethyl-4-piperidinone hydrochloride (TEMP) was used as... 1 O2 scavenger validation 1 The generation of O2, such as Figure 6 As shown in (A), a 565nm LED light (30mW / cm²) was used. 2 After irradiating a PBS solution containing SeR-NO (25 μM) / TEMP (100 mM) for 5 min, observations were made. 1The characteristic signal peak of the O2-TEMP adduct was not observed in either the SeR-NO / TEMP+ group (without light) or the TEMP+ group (under light) under the same conditions. Subsequently, 5,5-dimethyl-1-pyrrolidone-N-oxide (DMPO) was used as the O2 adduct. ·- Verification of O2 with NO·spin trapping agent ·- The generation of NO·, such as Figure 6 As shown in (B), a 565nm LED light (30mW / cm²) was used. 2 After irradiating a methanol solution of SeR-NO (1 mM) / DMPO (100 mM) for 10 min, O2 was observed. ·- The characteristic signal peaks of the NO· adduct with DMPO were observed, but no characteristic signal peaks were observed in either the SeR-NO / TEMP+ group (without illumination) or the TEMP+ group (under illumination) under the same conditions. These results indicate that SeR-NO simultaneously generates O2 under illumination. ·- , · NO and 1 O2.

[0056] The hypoxic environment within tumors typically limits the practical application of Type II photosensitizers. Therefore, the ability of SeR-NO to generate reactive oxygen species under normoxic and hypoxic conditions was further compared. Figure 7 As shown in (A) and (B), SeR-NO is produced under hypoxic conditions. 1 The O2 production capacity of SeR-NO is indeed significantly reduced compared to normoxic conditions; however, compared to normoxic conditions, the O2 produced by SeR-NO under hypoxic conditions is significantly higher. ·- Significantly increased. Importantly, since the release of NO· by SeR-NO under light is not limited by hypoxia, ONOO - The production of [the substance] also increases significantly. Therefore, the photodynamic effect of SeR-NO is not affected by cancer cell hypoxia, and it should have a very strong cancer cell killing ability under both normoxic and hypoxic conditions.

[0057] 4. SeR-NO-mediated photocatalytic oxidation of NADPH and reduction of Cyt c

[0058] Utilizing photocatalytic NADPH oxidation and Cyt c reduction to disrupt intracellular redox balance is of great significance in cancer treatment. Further testing was conducted on the effects of SeR-NO on NADPH / NAPD under normoxic and hypoxic conditions. + Photocatalysis. For example... Figure 8 As shown in (A) and (B), under normal oxygen conditions, a low-power 565nm LED light (10mW / cm²) was used. 2After irradiation with a PBS solution of SeR-NO (15 μM) / NADPH (240 μM), the characteristic absorption peak of NADPH at 339 nm decreased rapidly (over 4 minutes), while NAPD... + The characteristic absorption peak at 260 nm showed a significant increase, indicating that SeR-NO can photocatalyze the oxidation of NADPH, thereby altering the intracellular NADH / NAD ratio. + Balance. However, under hypoxic conditions, the rate and efficiency of NADPH oxidation are significantly reduced. This is because oxygen acts as an electron acceptor during SeR-NO catalysis of NADPH oxidation, and the rate and efficiency of NADPH oxidation also decrease when the oxygen concentration decreases.

[0059] Next, the electron acceptor Cyt c (60 μM) was added to the SeR-NO / NADPH PBS solution, followed by illumination (565 nm LED, 10 mW / cm²). 2 (Total 4 minutes), such as Figure 8 As shown in (C) and (D), the absorption peaks of Cyt c at 520 nm and 550 nm increase, indicating that SeR-NO catalyzes the reduction of Cyt c under illumination. Notably, the rate and efficiency of Cyt c reduction increase under hypoxic conditions. This is because Cyt c and oxygen both act as electron acceptors; compared to normoxic conditions, the oxygen concentration is lower in hypoxic environments, thus reducing competition for oxygen with Cyt c. These experimental results demonstrate that SeR-NO can catalyze the oxidation of NADPH and the reduction of Cyt c under illumination, and that this process is oxygen-independent.

[0060] 5. Study on the photodynamic therapy effect of photosensitizers at the cellular level

[0061] Given that the photosensitizer SeR-NO can efficiently generate reactive oxygen species / nitrogen under light irradiation and catalyze the oxidation of NADPH and the reduction of Cytc, the photodynamic therapy effect of this photosensitizer was further evaluated at the cellular level using laser scanning confocal microscopy (CLSM).

[0062] First, A549 cells pre-loaded with SeR-NO (5 μM, 30 min) were continuously irradiated with a 561 nm CLSM laser. Bright-field and fluorescence images were collected every 2 minutes for a total of 20 minutes. Figure 9 As shown in (A) and (B), the fluorescence signal in A549 cells gradually increased with prolonged irradiation time, and the cells simultaneously exhibited shrinkage and bubbling; however, unirradiated cells showed no changes. These experimental results indicate that SeR-NO readily penetrates the cell membrane and is rapidly activated under 561nm laser irradiation, simultaneously restoring its fluorescence and photodynamic activity.

[0063] To verify that cell death is caused by reactive oxygen species / nitrogen generated by SeR-NO, DCFH-DA, MCR-DMA, DHE, DAF-FM DA, and O71 were used to verify intracellular total ROS, 1 O2, O2 ·- ,NO·and ONOO - The generation of. For example... Figure 10 As shown, cells loaded with DCFH-DA / SeR-NO, MCR-DMA / SeR-NO, DHE / SeR-NO, DAF-FM DA / SeR-NO, or O71 / SeR-NO showed no fluorescence without light exposure, but exhibited strong fluorescence signals after irradiation with a 561 nm CLSM laser. These results indicate that SeR-NO simultaneously produces fluorescence in cells under light irradiation. 1 O2, NO·, O2 ·- and ONOO - .

[0064] To verify the photodynamic effect of SeR-NO, its phototoxicity and dark toxicity under normoxic and hypoxic conditions were tested using the CCK8 assay. A549 cells were incubated with different concentrations (0-0.5 μM) of SeR-NO for 1 hour, followed by 24 hours of culture in a cell culture incubator in the non-illuminated group, and then cultured under a 565 nm LED light source (20 mW / cm²). 2 After irradiation (15 min), the cells were placed in a cell culture incubator and cultured for another 24 hours. Figure 11 As shown, under both normoxic (21% O2) and hypoxic (2% O2) conditions, SeR-NO exhibits acceptable darkness in the 0-0.5 μM range. However, its phototoxicity gradually increases with increasing concentration, reaching a lower half-lethal concentration (EC50) under light irradiation. 50 The concentrations were 1.02 μM (21% O2) and 0.97 μM (2% O2), respectively. These experimental results demonstrate that SeR-NO possesses excellent photodynamic therapy efficacy that is independent of oxygen concentration, making it a photodynamic photosensitizer with potential clinical application value.

[0065] To further elucidate the mechanism of SeR-NO-induced A549 cell death, cell viability was determined when SeR-NO was co-incubated with different cell death inhibitors. For example... Figure 12 As shown, when A549 cells were co-incubated with the apoptosis inhibitor (Z-VAD-FMK), the necrosis inhibitor (Nec-1), and the autophagy inhibitor (3-MA), respectively, the A549 cells died rapidly under light, while the ferroptosis inhibitor Fer-1 increased the survival rate of A549 cells from 2.1% to 87.0%. Calcein-AM / PI staining experiments further validated this result. Figure 13As shown, A549 cells loaded with SeR-NO exhibited bright green fluorescence, indicating that SeR-NO has almost no dark toxicity; A549 cells loaded with SeR-NO showed bright red fluorescence after light exposure, indicating that SeR-NO has strong phototoxicity; while A549 cells loaded with SeR-NO / Fer-1 showed similar green fluorescence after light exposure as those loaded with SeR-NO alone, indicating that Fer-1 effectively inhibited A549 cell death. These results suggest that the mechanism of death induced by SeR-NO in A549 cells is ferroptosis.

[0066] In summary, this invention has developed an N-nitrosofunctionalized selenorhodamine-based photosensitizer, SeR-NO. SeR-NO can simultaneously generate a large amount of [unclear - possibly a specific substance or process] under 565nm LED light irradiation. 1 O2, NO· and O2 ·- At the same time, NO· and O2 are generated in the space. ·- Further cascade reactions occur to generate even more toxic ONOO. - Furthermore, SeR-NO can catalyze the oxidation of NADPH and the reduction of Cyt c under light. Therefore, SeR-NO exhibits significant phototoxicity and can induce A549 cell death via ferroptosis, with IC50 values ​​of 1.02 μM and 0.97 μM under normoxic (21% O2) and hypoxic (2% O2) conditions, respectively.

[0067] The foregoing has shown and described the main features and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0068] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A non-oxygen-dependent, multimodal seleno-rhodamine-based photodynamic photosensitizer, characterized in that, The structural formula of the photosensitizer is:

2. A method for preparing the non-oxygen-dependent multimode selenorodamine-based photodynamic photosensitizer according to claim 1, characterized in that, Includes the following steps: Step 1: Under nitrogen protection, 2-iodobenzoic acid and anhydrous THF were mixed and the resulting solution was cooled. Then, isopropyl magnesium chloride-lithium chloride solution was added dropwise to react. After heating, selenopyrrolidone THF solution was added dropwise to continue the reaction. The mixture was gradually restored to room temperature to continue the reaction. After the reaction was completed, the reaction solution was quenched under ice-water bath conditions, the solvent was evaporated, and the resulting solid was redissolved in a mixed solution of concentrated hydrochloric acid and methanol and the reaction was continued with stirring. The solvent was removed by vacuum distillation of the mixture, and the crude product was separated by chromatographic column to obtain 2-carboxyphenylselenopyrrolidone. Step 2: Dissolve 2-carboxyphenylselenodazole in dichloromethane, then add sodium hydroxide solution. The mixture is extracted, dried, filtered, and evaporated under reduced pressure to obtain a yellow oil. Dissolve the yellow oil in ultra-dry THF, add n-butyl nitrite, and stir to react. After the reaction is complete, evaporate the solvent under reduced pressure. The crude product is purified by silica gel column chromatography to obtain the photosensitizer.

3. The method for preparing a non-oxygen-dependent multimode selenorodamine-based photodynamic photosensitizer according to claim 2, characterized in that, In step 1, the molar ratio of 2-iodobenzoic acid, isopropyl magnesium chloride-lithium chloride, and selenopyronone is 10:20:

1.

4. The method for preparing a non-oxygen-dependent multimode selenorodamine-based photodynamic photosensitizer according to claim 2, characterized in that, In step 1, the temperature is cooled to -78°C, and the isopropyl magnesium chloride-lithium chloride solution is added dropwise for 30 minutes. After the temperature is raised to 0°C, the THF solution of selenopyronin is added dropwise for another 30 minutes. The temperature is then gradually restored to room temperature and the reaction continues for 48 hours.

5. The method for preparing a non-oxygen-dependent multimode selenorodamine-based photodynamic photosensitizer according to claim 2, characterized in that, In step 1, the volume ratio of concentrated hydrochloric acid to methanol is 1:

11.

6. The method for preparing a non-oxygen-dependent multimode selenorodamine-based photodynamic photosensitizer according to claim 2, characterized in that, In step 1, the developing solvent used in the chromatographic column is DCM and MeOH in a volume ratio of 20:

1.

7. The method for preparing a non-oxygen-dependent multimode selenorodamine-based photodynamic photosensitizer according to claim 2, characterized in that, In step 2, the molar ratio of 2-carboxyphenylselenomethoxymethyl sulfoxide to n-butyl nitrite is 1:

3.

8. The method for preparing a non-oxygen-dependent multimode selenorodamine-based photodynamic photosensitizer according to claim 2, characterized in that, In step 2, the sodium hydroxide solution has a mass fraction of 5%, the stirring temperature is 60°C, and the reaction time is overnight.

9. The method for preparing a non-oxygen-dependent multimode selenorodamine-based photodynamic photosensitizer according to claim 2, characterized in that, In step 2, the developing solvent used for silica gel column chromatography is PE and EA in a volume ratio of 10:

1.

10. The application of the non-oxygen-dependent multimodal selenorhodamine photodynamic photosensitizer of claim 1, characterized in that, Used to prepare reagents that kill cancer cells.

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