Non-oxygen-dependent multi-mode selenium rhodamine photodynamic photosensitizer as well as preparation and application thereof

Abstract

The invention belongs to the technical field of fluorescent probes, and particularly relates to a non-oxygen-dependent multi-mode selenium rhodamine photodynamic photosensitizer as well as preparation and application of the non-oxygen-dependent multi-mode selenium rhodamine photodynamic photosensitizer. In order to develop a non-oxygen-dependent and efficient photosensitizer, an N-nitroso functionalized selenium rhodamine photosensitizer SeR-NO is developed on the basis of a selenium rhodamine spiro ring'off-on 'mechanism, and the N-nitroso functionalized selenium rhodamine photosensitizer SeR-NO exists in a ring-closed lactone form in PBS (Phosphate Buffer Solution) and has extremely low cell dark toxicity and excellent cell permeability; under illumination, the photosensitizer not only can generate a large amount of ROS through a type I mechanism and a type II mechanism, but also can catalyze NADPH oxidation and Cyt c reduction in an oxygen-independent manner, so that SeR-NO has high phototoxicity, and can induce cell death in a ferroptosis manner in both normal oxygen (21% O2) and hypoxia (2% O2).

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a non-oxygen-dependent multi-mode selenorhodamine photodynamic photosensitizer and its preparation and application. Background Art

[0002] Photodynamic therapy (PDT) is a photochemical reaction-regulated treatment method that uses photosensitizers (PSs), light and oxygen (O 2 ) work together to produce reactive oxygen species (ROS), thereby achieving the purpose of treatment. Specifically, under light, PSs changes from the ground state (S 0 ) is excited to the singlet excited state (S 1 ), and then decays to the excited triplet state (T 1 ). Due to the long life, the generated T 1 The triplet-triplet energy transfer can directly transfer the energy of the photosensitizer to the surrounding O 2 , generating highly cytotoxic singlet oxygen ( 1 O 2 )(Type II mechanism); or cascade electron reactions or hydrogen abstraction reactions with adjacent biomolecules to generate superoxide (O 2 ·- ) and hydroxyl radicals (OH·) and other active intermediates (Type I mechanism), thereby causing apoptosis and / or necrosis of cancer cells.

[0003] Most of the photosensitizers reported or used clinically are type II photosensitizers, which are highly dependent on oxygen concentration. However, due to the rapid proliferation of tumor cells, the tumor is in an extremely hypoxic environment (pO 2 <5 mmHg), therefore, the application of type II photosensitizers in solid tumors is greatly limited. In contrast, type I photosensitizers have been shown to have low oxygen dependence. Recent studies have shown that some PSs can photocatalyze NADPH oxidation and thus change the intracellular NADH / NAD + Balance, eventually leading to cell death, this strategy is non-oxygen dependent. In addition, the combined use of PDT with other treatment methods (such as photothermal therapy, immunotherapy, gas therapy) can significantly improve the therapeutic effect of tumors and improve the many defects of single PDT (such as tumor resistance, proliferation, metastasis, and high oxygen dependence, etc.), providing new ideas for the development of non-oxygen dependent and efficient photosensitizers. Summary of the invention

[0004] Selenorhodamine and its derivatives are natural photodynamic photosensitizers, which is mainly attributed to the ISC process promoted by selenium atoms in their molecular structures. However, as a member of the rhodamine dye family, photodynamic photosensitizers developed based on the "off-on" mechanism of the 2-carboxyphenylselenorhodamine spirocyclic ring are rarely reported. Based on this, the present invention develops an N-nitroso-functionalized selenorhodamine photosensitizer SeR-NO, which exists in the form of a closed-ring lactone in PBS and has extremely low dark cytotoxicity and excellent cell permeability; under light, it can not only produce a large amount of ROS through type I and type II mechanisms, but also catalyze NADPH oxidation and Cyt c reduction in an oxygen-independent manner. Therefore, SeR-NO has great phototoxicity and is highly phototoxic in normoxia (21% O 2 ) and hypoxia (2% O 2 ) can induce cell death through ferroptosis.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a non-oxygen-dependent multi-mode selenorhodamine photodynamic photosensitizer, the structural formula of which is:

[0007]

[0008] The present invention also provides a method for preparing an oxygen-independent multi-mode selenorhodamine photodynamic photosensitizer, comprising the following steps:

[0009]

[0010] Step 1, under nitrogen protection, 2-iodobenzoic acid and anhydrous THF are mixed, and the resulting solution is cooled, and then an isopropyl magnesium chloride-lithium chloride solution is added dropwise to react, and after heating, a THF solution of selenopyroxine is added dropwise to continue the reaction, and the temperature is gradually restored to room temperature to continue the reaction. After the reaction is completed, the reaction solution is quenched in an ice-water bath, the solvent is spin-dried, and the obtained solid is redissolved in a mixed solution of concentrated hydrochloric acid and methanol and the reaction is continued by stirring, the mixture is subjected to reduced pressure distillation to remove the solvent, and the crude product is separated by a chromatographic column to obtain 2-carboxyphenylselenodamine;

[0011] Step 2, dissolving 2-carboxyphenylselenodamine in dichloromethane, then adding sodium hydroxide solution, extracting, drying, filtering and evaporating the mixture under reduced pressure to obtain a yellow oil, dissolving the yellow oil in ultra-dry THF, adding n-butyl nitrite to react with stirring, after the reaction is completed, evaporating the solvent under reduced pressure, and separating and purifying the crude product by silica gel column chromatography to obtain the photosensitizer.

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

[0013] Furthermore, in step 1, the reaction mixture is cooled to -78°C, and an isopropylmagnesium chloride-lithium chloride solution is added dropwise for 30 minutes. After the temperature is raised to 0°C, a THF solution of selenopyroxine is added dropwise for a further 30 minutes, and the temperature is gradually restored to room temperature for a further 48 hours.

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

[0015] Furthermore, it is characterized in that the developing solvents used for the chromatographic column in step 1 are DCM and MeOH, and the volume ratio thereof is 20:1.

[0016] Furthermore, it is characterized in that in step 2, the molar ratio of 2-carboxyphenylselenodamine to n-butyl nitrite is 1:3.

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

[0018] Furthermore, the developing solvents used for the silica gel column chromatography in step 2 are PE and EA, and the volume ratio thereof is 10:1.

[0019] The present invention also provides an application of a non-oxygen-dependent multi-mode selenorhodamine photodynamic photosensitizer for killing cancer cells under the irradiation of a 561nm LED light source.

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

[0021] The present invention develops a photodynamic photosensitizer SeR-NO based on the "off-on" mechanism of selenorhodamine spirocyclic ring. Specifically, 2-iodobenzoic acid is reacted with isopropylmagnesium chloride-lithium chloride solution, and the obtained Grignard reagent is further subjected to nucleophilic addition / elimination reaction with selenopyrrolidone to obtain 2-carboxyphenylselenarhodamine with a yield of 69.6%. 2-carboxyphenylselenarhodamine is further reacted with n-butyl nitrite to finally obtain the photosensitizer SeR-NO with a yield of 77%.

[0022] Under 565 nm LED light irradiation, SeR-NO rapidly releases NO and photosensitizer SeR, and the released SeR simultaneously and spatially produces a large amount of O through type I and type II mechanisms. 2 ·- and 1 O 2 , O 2 ·- SeR-NO can further catalyze NADPH oxidation and Cyt c reduction in an oxygen-independent manner under light.

[0023] CCK8 experiments showed that SeR-NO could induce A549 cell death through ferroptosis. 2 ) and hypoxia (2% O 2 ) were 1.02 μM and 0.97 μM, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 SeR-NO (2 μM) solution in PBS was illuminated with different wavelengths of LED light (10 mW / cm 2 ) fluorescence spectrum changes under irradiation.

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

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

[0027] Figure 4 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA) was irradiated with laser (565nm, 10mW / cm 2 ) under irradiation, where (A) is ABDA and SeR-NO, and (B) is ABDA and RB.

[0028] Figure 5 (A) shows DHR 123 under laser (565nm, 10mW / cm 2 ) is the fluorescence spectrum change diagram of superoxide produced by the photosensitizer under the irradiation of laser (565nm, 10mW / cm 2 ) Fluorescence spectrum changes of peroxynitrite anions produced by the captured photosensitizer under irradiation.

[0029] Figure 6 (A) and (B) are captured by TEMP (in PBS) and DMPO (in methanol), respectively. 1 O 2 and O 2 ·- EPR spectrum of / NO·.

[0030] Figure 7 (A) and (B) show the production of SeR-NO in oxygen-saturated PBS and nitrogen-saturated PBS, respectively. 1 O 2 ,,O 2 ·- and ONOO -situation.

[0031] Figure 8 (A) and (B) are oxygen-saturated PBS solution of SeR-NO (15 μM) / NADPH (240 μM) and nitrogen-saturated PBS under light (565 nm LED, 10 mW / cm 2 ) UV-visible absorption spectrum change diagram; (C) and (D) are oxygen-saturated PBS solution of SeR-NO (15μM) / NADPH (240μM) / Cyt c (60μM) and nitrogen-saturated PBS under light (565nm LED, 10mW / cm 2 )'s UV-visible absorption spectrum changes.

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

[0033] Fig.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 without illumination or illumination. The collection ranges are: 500-550nm (λex=488nm), 650-750nm (λex=633nm), 570-630nm (λex=561nm), 500-540nm (λex=488nm) and 500-550nm (λex=488nm), scale bar: 20μm.

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

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

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

[0037] In order to further illustrate the technical solution of the present invention, the present invention is further described below through embodiments.

[0038] Example 1

[0039] A non-oxygen-dependent multi-mode selenorhodamine photodynamic photosensitizer, the structural formula of which is:

[0040]

[0041] A method for preparing a non-oxygen-dependent multi-mode selenorhodamine photodynamic photosensitizer comprises the following steps:

[0042] Step 1, add 2-iodobenzoic acid (719.2 mg, 2.9 mmol) to a dry round-bottom flask, add anhydrous THF (2.0 mL) under nitrogen protection, and cool the resulting solution to -78 ° C, then add isopropylmagnesium chloride-lithium chloride solution (1.3M, 4.5 mL, 5.8 mmol) dropwise. After the reaction solution reacts at -78 ° C for 30 minutes, it is warmed to 0 ° C, and then a THF solution of selenopyroxithone (100 mg, 0.29 mmol) is added dropwise. After the reaction solution reacts at 0 ° C for 30 minutes, it gradually returns to room temperature, and the reaction solution reacts at room temperature for 48 hours. After the reaction is completed, the reaction solution is quenched with methanol (5.0 mL) under ice-water bath conditions, and after the solvent is spin-dried, the solid is redissolved in a mixed solution of concentrated hydrochloric acid and methanol (5.0 mL, volume ratio of 1:11), and stirring is continued for 10 minutes. The mixture was distilled under reduced pressure to remove the solvent, and the crude product was separated by chromatography (DCM / MeOH=20 / 1, V / V) to give 2-carboxyphenylselenodamine as a blue-purple solid (98.0 mg, 69.6%).

[0043] 1 H NMR (600 Hz, CDCl 3 )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=7.2Hz,1H),7.02(d,J=9.0Hz,2H),6.91(d,J=2.4Hz,2H),7.02(dd,J 1 =8.4Hz,J 2 =2.4 Hz, 2H), 2.96 (s, 12H); 13 C NMR (150 MHz, CDCl 3)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-carboxyphenylselenodamine (100.0 mg, 0.2 mmol) was dissolved in DCM (10.0 mL), followed by addition of 5% sodium hydroxide (NaOH) solution (20.0 mL), the mixture was extracted 3 times with DCM, and the organic phase was washed with anhydrous Na 2 SO 4 Dry, filter, and evaporate under reduced pressure to obtain a yellow oil. The yellow oil was dissolved in ultra-dry THF (15.0 mL), and n-butyl nitrite (n-BuONO) (64.9 mg, 0.6 mmol) was added, and the mixture was stirred at 60°C overnight. After the reaction was completed, the solvent was evaporated under reduced pressure, and the crude product was separated and purified by silica gel column chromatography (PE / EA=10 / 1, V / V) to obtain SeR-NO as a white solid (74.7 mg, 77%).

[0045] 1 H NMR (600 MHz, DMSO-d 6 )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=8.7,2.3 Hz,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 (150 MHz, DMSO-d 6 )δ169.53,153.02,150.64,142.23,135.82,133.41,130.91,130.73,129.35,128.01,126 .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] Chromatographically pure CH 3 CN was used to prepare the stock solution of SeR-NO (2 mM), which was diluted to the required concentration with PBS (10 mM, pH = 7.4) during the test. Ultrapure water was used to prepare 9,10-anthracenediyl-di(methylene)dimalonic acid (ABDA, 5 mM), NADPH (50 mM), and Cytc (5 mM). Chromatographically pure DMF was used to prepare DHR123 (2 mM) and DHE (2 mM) solutions. Sterile DMSO was used to prepare cell death inhibitors (10 mM), including necrosis inhibitors (Necrostatin-1, Nec-1), ferroptosis inhibitors (Ferrostatin-1, Fer-1), apoptosis inhibitors (Z-VAD-FMK), and autophagy inhibitors 3-methyladenine (3-MA).

[0049] 2. Study on the in vitro performance of photosensitizers

[0050] First, the ability of SeR-NO (2 μM) to be activated by light was investigated in PBS using a fluorescence spectrophotometer. Figure 1 As shown, SeR-NO exists in the form of a ring-closed lactone in PBS. When the ions are illuminated at 375 nm, 450 nm, 505 nm, 565 nm and 625 nm (10 mW / cm 2 ) LED light source irradiated SeR-NO, the fluorescence at 595nm was significantly enhanced, and the fluorescence intensity reached equilibrium in about 30s, and the color of the solution changed from colorless to purple. HPLC-MS results confirmed that SeR-NO generated the de-NO product SeR( Figure 2 ). Then, Griess reagent was used to verify that SeR-NO (2 μM) could release NO (2 μM) with an efficiency of about 100% under light irradiation ( Figure 3 ).

[0051] Since the maximum absorption wavelength of the photosensitizer SeR is about 565nm, a 565nm LED light source is selected as the final excitation light source. That is to say, under the irradiation of 565nm LED light, SeR-NO can quickly release NO· and photosensitizer SeR, and the released SeR immediately produces large reactive oxygen species (ROS) under light, thus achieving the simultaneous release of NO· and other ROS.

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

[0053] First, take ABDA as 1 O 2The capture agent, Rose Bengal (RB) was used as a reference to test the production of SeR-NO in PBS 1 O 2 Such as Figure 4 As shown in (A), a 565nm LED light source (10mW / cm 2 When ABDA / SeR-NO solution (or ABDA / RB solution) was continuously irradiated with light for 10 seconds, the absorption peak at 565nm gradually increased, and the absorbance value of ABDA at 380nm decreased rapidly. The experimental results showed that SeR-NO released the photosensitizer SeR under light and simultaneously produced a large amount of 1 O 2 , the singlet oxygen generation efficiency is significantly stronger than that of RB ( Figure 4 (B)).

[0054] Subsequently, the commercial fluorescent probe DHR 123 was used to test the production of O by SeR-NO in PBS. 2 ·- Such as Figure 5 As shown in (A), when using a 565nm LED light source (565nm LED, 10mW / cm 2 When the DHR 123 / SeR-NO solution was continuously irradiated with light (10 s interval), the fluorescence intensity at 530 nm gradually increased, indicating that SeR-NO can produce a large amount of O under light. 2 ·- It is noteworthy that SeR-NO releases O 2 ·- The cascade reaction between SeR-NO and NO· can further generate more toxic ONOO-. Next, the commercial fluorescent probe O71 was used to test the production of ONOO by SeR-NO under light. - Such as Figure 5 As shown in (B), when using a 565nm LED light source (565nm LED, 10mW / cm 2 When the O71 / SeR-NO solution was continuously irradiated with 10 s interval, the fluorescence at 530 nm was significantly enhanced, confirming that ONOO - The above experimental results confirm that SeR-NO can produce a large amount of 1 O 2 and O 2 ·- , O 2 ·- It can further react with NO· released in the same space and time to generate more toxic ONOO-.

[0055] Furthermore, EPR spectroscopy was used to determine the free radicals generated by SeR-NO under light. First, 2,2,6,6-tetramethyl-4-piperidone hydrochloride (TEMP) was used as 1 O 2 Capture agent validation 1 O 2 The production of Figure 6 As shown in (A), 565nm LED light (30mW / cm 2 After irradiation of SeR-NO (25 μM) / TEMP (100 mM) PBS solution for 5 min, it was observed 1 O 2 The characteristic signal peak of TEMP adduct was not observed in SeR-NO / TEMP+no-illumination group and TEMP+illumination group under the same conditions. 2 ·- and NO· Spin trapping agent validation O 2 ·- and NO·, such as Figure 6 As shown in (B), 565nm LED light (30mW / cm 2 After irradiation of SeR-NO (1 mM) / DMPO (100 mM) methanol solution for 10 min, O 2 ·- The characteristic signal peaks of NO· and DMPO adducts were observed in the SeR-NO / TEMP+no-illumination group and the TEMP+illumination group under the same conditions. The above results indicate that SeR-NO simultaneously generates O 2 ·- , · NO and 1 O 2 .

[0056] The hypoxic environment in tumors usually limits the practical application of Type II photosensitizers, so we further compared the ability of SeR-NO to produce reactive oxygen species under normoxic and hypoxic conditions. Figure 7 As shown in (A) and (B), SeR-NO is produced under hypoxic conditions. 1 O 2 The ability of SeR-NO to produce O under hypoxia was significantly reduced compared with normoxic conditions; however, compared with normoxic conditions, the amount of O produced by SeR-NO under hypoxia was 2 ·- Importantly, since the process of SeR-NO releasing NO under light is not restricted by hypoxia, ONOO -Therefore, the photodynamic effect of SeR-NO will not be affected by hypoxia of cancer cells, and it should have a strong ability to kill cancer cells under normoxic or hypoxic conditions.

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

[0058] Disruption of intracellular redox balance by photocatalytic NADPH oxidation and Cyt c reduction is of great significance in cancer treatment. + Photocatalytic effect. Figure 8 As shown in (A) and (B), under normoxic conditions, low-power 565nm LED light (10mW / cm 2 After irradiation of SeR-NO (15 μM) / NADPH (240 μM) PBS solution, the characteristic absorption peak of NADPH at 339 nm decreased rapidly (4 min), while NADPH + The characteristic absorption peak at 260 nm increased significantly, which means that SeR-NO can photocatalyze NADPH oxidation and thus change the intracellular NADH / NAD + However, under hypoxic conditions, the rate and efficiency of NADPH oxidation are significantly reduced. This is because when SeR-NO catalyzes NADPH oxidation, oxygen serves as an electron acceptor. When the oxygen concentration decreases, the rate and efficiency of NADPH oxidation will also decrease.

[0059] Next, the electron acceptor Cyt c (60 μM) was added to the SeR-NO / NADPH PBS solution and then illuminated (565 nm LED, 10 mW / cm 2 , 4 min in total), such as Figure 8 As shown in (C) and (D), the absorption peaks of Cyt c at 520nm and 550nm increase, indicating that SeR-NO catalyzes the reduction of Cyt c under light. It is worth noting that the rate and efficiency of Cyt c reduction increase under hypoxia. This is because Cyt c and oxygen act as electron acceptors. Compared with normoxia, the oxygen concentration in hypoxia is lower, and the competition for Cyt c with oxygen is weakened. The above experimental results show that SeR-NO can catalyze NADPH oxidation and Cyt c reduction under light, and the process is non-oxygen dependent.

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

[0061] In view of the characteristics of the photosensitizer SeR-NO that it can efficiently produce reactive oxygen and nitrogen under light, and catalyze NADPH oxidation and Cytc reduction, laser scanning confocal microscopy (CLSM) was used to further evaluate the photodynamic therapy effect of this photosensitizer at the cellular level.

[0062] First, A549 cells pre-loaded with SeR-NO (5 μM, 30 min) were continuously irradiated with 561 nm CLSM laser, and bright field images and fluorescence images were collected every 2 min for a total of 20 min. Fig. 9 As shown in (A) and (B), as the irradiation time increases, the fluorescence signal in A549 cells gradually increases, and at the same time, the cells shrink and blister; however, there is no change in the unirradiated cells. The above experimental results show that SeR-NO can easily penetrate the cell membrane and is quickly activated under 561nm laser irradiation, while restoring fluorescence and photodynamic activity.

[0063] To verify that cell death was caused by reactive oxygen and nitrogen generated by SeR-NO, DCFH-DA, MCR-DMA, DHE, DAF-FM DA and O71 were used to verify the total intracellular ROS, 1 O 2 , O 2 ·- ,NO·and ONOO - The production of. Fig.10 As shown in the figure, 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 when not irradiated with light, but after irradiation with 561nm CLSM laser, strong fluorescence signals appeared. These results indicate that SeR-NO simultaneously produces 1 O 2 、NO·、O 2 ·- and ONOO - .

[0064] To verify the photodynamic effect of SeR-NO, the phototoxicity and dark toxicity of SeR-NO under normoxic and hypoxic conditions were tested by CCK8 assay. A549 cells were incubated with SeR-NO at different concentrations (0-0.5 μM) for 1 hour, and then the non-illumination group was placed in a cell culture incubator for 24 hours. The illumination group was illuminated with a 565 nm LED light source (20 mW / cm 2 , 15min) and then placed in a cell culture incubator for 24 hours. Fig.11 As shown, in normoxia (21% O 2 ) and hypoxia (2% O 2) condition, SeR-NO has acceptable dark resistance in the range of 0-0.5 μM, however, its phototoxicity gradually increases with the increase of concentration, and the half lethal concentration (EC 50 ) were 1.02 μM (21% O 2 ) and 0.97 μM (2% O 2 ). The above experimental results show that SeR-NO has excellent photodynamic therapy effect and the therapeutic effect is not dependent on oxygen concentration. It is a photodynamic photosensitizer with potential clinical application value.

[0065] To further clarify the death mode of A549 cells induced by SeR-NO, the cell survival rate was measured when SeR-NO was co-incubated with different death inhibitors. Fig.12 As shown in Figure 2, when A549 cells were co-incubated with apoptosis inhibitors (Z-VAD-FMK), necrosis inhibitors (Nec-1), and autophagy inhibitors (3-MA), 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 verified this result, as shown in Figure 2. Fig.13 As shown, A549 cells loaded with SeR-NO showed bright green fluorescence, indicating that SeR-NO had almost no dark toxicity; A549 cells loaded with SeR-NO showed bright red fluorescence after illumination, indicating that SeR-NO had strong phototoxicity; and A549 cells loaded with SeR-NO / Fer-1 showed similar green fluorescence to that loaded with SeR-NO alone after illumination, indicating that Fer-1 effectively inhibited the death of A549 cells. The above results indicate that the death mode of A549 cells induced by SeR-NO is ferroptosis.

[0066] In summary, the present invention has developed an N-nitroso-functionalized selenorhodamine photosensitizer SeR-NO. SeR-NO can produce a large amount of 1 O 2 , NO. and O 2 ·- , NO· and O 2 ·- Further cascade reactions occur to generate more toxic ONOO - Moreover, SeR-NO can also catalyze NADPH oxidation and Cyt c reduction under light. Therefore, SeR-NO has strong phototoxicity and can induce A549 cell death through ferroptosis. 2 ) and hypoxia (2% O 2) were 1.02 μM and 0.97 μM, respectively.

[0067] The above shows and describes the main features and advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes that fall within the meaning and scope of the equivalent elements of the claims are included in the present invention.

[0068] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A non-oxygen-dependent multi-mode selenorhodamine photodynamic photosensitizer, characterized in that: The structural formula of the photosensitizer is:

2. A method for preparing the oxygen-independent multi-mode selenorhodamine photodynamic photosensitizer according to claim 1, characterized in that: The following steps are involved: Step 1, under nitrogen protection, 2-iodobenzoic acid and anhydrous THF are mixed, and the resulting solution is cooled, and then an isopropyl magnesium chloride-lithium chloride solution is added dropwise to react, and after heating, a THF solution of selenopyroxine is added dropwise to continue the reaction, and the temperature is gradually restored to room temperature to continue the reaction. After the reaction is completed, the reaction solution is quenched in an ice-water bath, the solvent is spin-dried, and the obtained solid is redissolved in a mixed solution of concentrated hydrochloric acid and methanol and the reaction is continued by stirring, the mixture is subjected to reduced pressure distillation to remove the solvent, and the crude product is separated by a chromatographic column to obtain 2-carboxyphenylselenodamine; Step 2, dissolving 2-carboxyphenylselenodamine in dichloromethane, then adding sodium hydroxide solution, extracting, drying, filtering and evaporating the mixture under reduced pressure to obtain a yellow oil, dissolving the yellow oil in ultra-dry THF, adding n-butyl nitrite to react with stirring, after the reaction is completed, evaporating the solvent under reduced pressure, and separating and purifying the crude product by silica gel column chromatography to obtain the photosensitizer.

3. The method for preparing a non-oxygen-dependent multi-mode selenorhodamine photodynamic photosensitizer according to claim 2, characterized in that: In the step 1, the molar ratio of 2-iodobenzoic acid, isopropylmagnesium chloride-lithium chloride and selenopyronine is 10:20:

1.

4. The method for preparing a non-oxygen-dependent multi-mode selenorhodamine photodynamic photosensitizer according to claim 2, characterized in that: In the step 1, the reaction is cooled to -78°C, and isopropylmagnesium chloride-lithium chloride solution is added dropwise for 30 minutes. After the temperature is raised to 0°C, a THF solution of selenopyroxine is added dropwise for a further reaction of 30 minutes, and the temperature is gradually restored to room temperature for a further reaction of 48 hours.

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

11.

6. The method for preparing a non-oxygen-dependent multi-mode selenorhodamine photodynamic photosensitizer according to claim 2, characterized in that: The developing solvents used for the chromatographic column in step 1 are DCM and MeOH in a volume ratio of 20:

1.

7. The method for preparing a non-oxygen-dependent multi-mode selenorhodamine photodynamic photosensitizer according to claim 2, characterized in that: In the step 2, the molar ratio of 2-carboxyphenylselenodamine to n-butyl nitrite is 1:

3.

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

9. The method for preparing a non-oxygen-dependent multi-mode selenorhodamine photodynamic photosensitizer according to claim 2, characterized in that: The developing solvents used for the silica gel column chromatography in step 2 are PE and EA, and the volume ratio thereof is 10:

1.

10. A use of the oxygen-independent multi-mode selenorhodamine photodynamic photosensitizer according to claim 1, characterized in that: Used to prepare reagents for killing cancer cells.

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