A photosensitizer based on photoactivated RNA labeling using phenothiazine, and its preparation method and application

Abstract

The present invention discloses a phenothiazine-based photoactivated RNA labeling photosensitizer, and its preparation method and application. The structure of the photosensitizer dye is shown in Formula I. The photosensitizer dye is based on phenothiazine photosensitizer dyes and is obtained by non-covalently linking furan fragments to obtain FNBS. The photosensitizer generates reactive oxygen species under light, which can oxidize furan to cause it to open its ring and then combine with nucleic acids in the cytoplasm to achieve the purpose of labeling. At the same time, it induces nucleic acid damage under light to cause cell pyroptosis, thereby achieving photoimmunotherapy. The photosensitizer has a strong killing ability against tumor cells. The anti-tumor effect of the photosensitizer dye includes the two parts of photosensitizer dye generating reactive oxygen species to kill tumor cells and cell death caused by destroying nucleic acids; inducing cell pyroptosis, causing cell immunogenic death. The advantage of the present invention is that the photosensitizer can not only achieve photodynamic therapy but also cause cell pyroptosis, thereby achieving photoimmunotherapy and improving the efficacy of tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and in particular relates to a method for preparing a photosensitizing dye in optical imaging and photodynamic therapy and its application. Background Art

[0002] Photodynamic therapy (PDT) is widely used in anti-tumor phototherapy due to its non-invasive nature, lack of drug resistance, and low toxicity. The three key elements of PDT are light, oxygen, and a photosensitizer. The wavelength of light determines its penetration depth, oxygen determines its efficiency, and the photosensitizing ability of the photosensitizer has been a limiting factor in its development. Of these three elements, the photosensitizer is central. Ideal photosensitizers for PDT require near-infrared absorption and emission, low oxygen dependency, and high photosensitization efficiency. PDT can be categorized into three types based on their mechanism of action: Type I, in which the excited state of the photosensitizer generates free radicals through electron transfer. This mechanism is less oxygen-dependent and exhibits higher cytotoxicity than singlet oxygen. Type II, in which the excited state of the photosensitizer converts ambient oxygen to singlet oxygen through energy transfer. However, the hypoxic environment of tumors limits the application of this type of photosensitizer in phototherapy. Type III, in which the excited state of the photosensitizer directly interacts with biomass molecules, damaging them and killing tumor cells. This mechanism is independent of oxygen and holds great promise for anti-tumor phototherapy. So far, although many photosensitizers based on different mechanisms have been developed, there are still many problems. For example, although cyanine photosensitizers have good photosensitization efficiency, they have poor photostability. Phthalocyanine and fluoroboron dipyrrole photosensitizers have poor aggregation quenching and biocompatibility due to their large conjugated plane. In contrast, phenothiazine photosensitizers have excellent water solubility and biocompatibility due to their cationic structure and small molecular weight, and they have both type I and type II phototherapy mechanisms and have attracted much attention. For example, methylene blue is a classic phenothiazine dye that is widely used in tumor photodiagnosis and treatment. In summary, further improving the photosensitization efficiency of photosensitizers, improving the photodynamic therapy effect in hypoxic environments, and developing photosensitizers with excellent biocompatibility and stability are still issues that need to be solved urgently.

[0003] In addition, the anti-tumor efficacy of photosensitizers is not only related to the photosensitization efficiency of the photosensitizers, but also to the distribution and binding sites of the photosensitizers in the cells. As we all know, the range of action of reactive oxygen species is limited. If the photosensitizers are distributed in the "energy factories" (mitochondria) and "digestive workshops" (lysosomes) of cells or bind to nucleic acids and specific proteins to destroy cell homeostasis, it will be beneficial to promote cell death and enhance the immune response. Compared with single phototherapy, photoimmunotherapy not only has a better inhibitory effect on in situ tumors, but can also inhibit tumor metastasis and recurrence. Currently, there have been many reports on subcellular organelle-targeted photosensitizers, but few reports on nucleic acid-targeted photosensitizers, especially photosensitizers targeting cytoplasmic nucleic acids, which can avoid genotoxicity.

[0004] While photodynamic therapy (PDT) has shown promise in the fight against tumors, existing photosensitizers are limited in their ability to precisely target key sites within cells. Most photosensitizers fail to effectively target nucleic acids in the cytoplasm, or targeting nucleic acids in the nucleus can cause genotoxicity, limiting the efficacy of PDT or posing unknown risks.

[0005] The tumor microenvironment, especially its hypoxic state, is a major challenge facing photodynamic therapy. Existing photosensitizers often rely on oxygen to produce reactive oxygen species, but in the hypoxic tumor environment, this dependence significantly reduces the therapeutic effect.

[0006] Based on the above problems, the development of cytoplasmic nucleic acid-targeted photosensitizer dyes is of great significance. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a preparation method and application of a phenothiazine-based photosensitizer. The photosensitizer can bind to RNA through light activation to achieve RNA labeling and anti-tumor phototherapy. In addition, it can serve as a stimulus source to activate the body's immunity and inhibit tumor metastasis and recurrence.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] The first aspect of the present invention provides a photoactivated RNA-labeled phenothiazine photosensitizer having a structure shown in the following formula I:

[0010]

[0011] The R1 is selected from a nitrogen-containing functional group; further preferably, the nitrogen-containing functional group is selected from one of dimethylamino, diethylamino, aziridine, azetidine, aziridine, and aziridine.

[0012] Further preferably, the photosensitizer is selected from one of C1, C2, C3, C4, C5, and C6;

[0013]

[0014] The compound has the following characteristics;

[0015] The main absorption peak is located at about 640nm±20nm, and the characteristic emission peak is located at 660nm±20nm in the near-infrared region;

[0016] Under light conditions, the singlet oxygen yield was as high as 0.32 ± 0.05;

[0017] RNA binding capacity, photosensitizer at 1 μM concentration and 660 nm LED light (2 mW / cm 2 , 10 min) photosensitizer can be covalently bound to RNA;

[0018] It is quickly taken up by target cells, and the cell survival rate of the group with only photosensitizer added is higher than 90%, indicating that it has no obvious toxicity to cells under no light conditions;

[0019] Under extremely low light dose (2mW / cm 2 , 10min), the cell survival rate dropped to 20%±5%, and at the same time, cell pyroptosis was triggered to induce immunogenic death and enhance the body's immune response.

[0020] The photosensitizing dye molecule FNBS is formed by connecting a phenothiazine photosensitizing dye platform containing a fatty acid group and a furan structure through an amidation reaction.

[0021] The second aspect of the present invention provides a method for preparing the photosensitizer according to the first aspect, comprising the following steps:

[0022]

[0023] The specific preparation steps are as follows:

[0024] 1) In an aqueous solution of aluminum sulfate, an aniline derivative is mixed with sodium thiosulfate and zinc chloride, and an aqueous solution of potassium dichromate is added. The mixture is stirred at low temperature, and the precipitate is filtered and washed. The crude product is refluxed in methanol and filtered to obtain intermediate 2;

[0025] 2) 1-amino-5,6,7,8-tetrahydronaphthylamine was refluxed with 3-bromopropionic acid and potassium carbonate in acetonitrile, and the intermediate 3 was obtained by column chromatography;

[0026] 3) Intermediate 2 was stirred with 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and triethylamine in dichloromethane at room temperature, furan-2-methylamine was added, and intermediate 4 was obtained by column chromatography.

[0027] 4) Intermediate 2 and intermediate 4 were refluxed in methanol under the catalysis of silver carbonate, and the target compound FNBS was separated by column chromatography.

[0028] For the technical solution described above, it is further preferred that in step 1), the molar ratio of 1-amino-5,6,7,8-tetralin, 3-bromopropionic acid and potassium carbonate is (0.8-1.2):(1-2):(1-2).

[0029] For the technical solution described above, it is further preferred that the eluent for the column chromatography separation in steps 2, 3, and 4 is a mixture of dichloromethane and methanol in a volume ratio of 20-100:1.

[0030] For the technical solution described above, it is further preferred that the aniline derivative in step 1) is preferably N,N-dimethyl-p-phenylenediamine, 4-aziridine-based aniline, 4-azetidinyl aniline, 4-azacyclopentyl aniline, 4-azacyclohexyl aniline, etc.; more preferably N,N-diethyl-p-phenylenediamine.

[0031] For the technical solution described above, it is further preferred that the molar amount of aluminum sulfate in step 1) is preferably 1-3 equivalents; more preferably 2 equivalents.

[0032] For the technical solution described above, it is further preferred that the molar ratio of the intermediate 1, 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, triethylamine and furan-2-methylamine in the step 2) is 1:(1-2):(1-2):(1-2):(1-3), and the solvent used for the extraction is dichloromethane and water in a volume ratio of 1:1-2;

[0033] For the technical solution described above, it is further preferred that in step 3), the molar ratio of the aniline derivative, aluminum sulfate, sodium thiosulfate, zinc chloride and potassium dichromate is 1:(1-2):(1-2):(0.1-0.5).

[0034] For the technical solution described above, it is further preferred that the organic solvent in step 1) is acetone.

[0035] For the technical solution described above, it is further preferred that the column chromatography separation solvent in step 3) is petroleum ether and ethyl acetate (10 / 1-1 / 1, v / v).

[0036] For the technical solution described above, it is further preferred that the molar ratio of intermediate 2 to intermediate 4 in step 4) is 1:1-3.

[0037] The third aspect of the present invention is to protect the use of the compound in the preparation of drugs for non-disease diagnosis and treatment, particularly in the field of inhibiting distal tumor growth and achieving photoimmunotherapy effects, wherein the photosensitizer promotes immune response and cell-selective effects through its unique photodynamic properties.

[0038] Specifically, the application directions of the photosensitizer include:

[0039] Preparation of drugs for photodynamic therapy (PDT); generating reactive oxygen species through light activation to achieve precise killing of target cells; at extremely low drug doses and light doses (2mW / cm 2 The killing efficiency of target cells reached 90% ± 5% at the concentration of 320nM after 10min of illumination.

[0040] Prepare reagents for RNA labeling and research, used to label RNA to facilitate the study of the role of RNA in cell metabolism and disease development.

[0041] Prepare drugs for tumor immunotherapy, which enhance the body's immune response to target cells by inducing immunogenic death; the dendritic cell immune response is increased by 100% ± 20%, and the T cell immune response efficiency is increased by 200% ± 20%.

[0042] Prepare reagents for pyroptosis research as research tools to explore the mechanisms of pyroptosis and understand its role in disease progression.

[0043] More preferably, the compound is used in the preparation of a medicament for photodynamic therapy and / or immunotherapy, where the photosensitizer can bind to RNA via light activation, thereby achieving RNA labeling and regulation, and promoting anti-tumor effects. More specifically, the compound specifically binds to RNA within tumor cells via light activation, achieving precise labeling and efficient destruction of tumor cells, while also enhancing the body's immune response to tumors by activating T lymphocytes, significantly reducing the risk of tumor metastasis and recurrence.

[0044] Advantages and beneficial effects of the present invention:

[0045] Given its excellent therapeutic efficacy and innovative mechanism, the photosensitizer of this invention is expected to become a powerful tool for clinical anti-tumor treatment. While promoting clinical applications, the present invention also prioritizes safety considerations. Through rigorous experimental verification of the photosensitizer's biocompatibility and safety in animal models, it ensures a safe margin for human use.

[0046] Compared with existing photodynamic therapy and photoimmunotherapy, the photosensitizer of this invention demonstrates significant advantages in terms of therapeutic efficacy, scope of application, and side effect management. In particular, its unique mechanism of action and high efficacy offer new hope for patients in the treatment of difficult-to-treat metastatic tumors.

[0047] This invention not only breaks new ground in the current field of photodynamic therapy but also provides a rich research direction for future photoimmunotherapy. As we further explore the mechanism of action of FNBS, we expect it to demonstrate greater potential in treating more types of tumors and further broaden its clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 is the H NMR spectrum of the target compound FNBS;

[0049] Figure 2 is the C NMR spectrum of the target compound FNBS;

[0050] Figure 3 This is the high-resolution mass spectrum of the target compound FNBS;

[0051] Figure 4 Spectra of the target compound FNBS in different solvents. (a) Absorption spectrum, (b) Fluorescence spectrum;

[0052] Figure 5 The singlet oxygen production capacity of the target compound FNBS and the comparison molecule NBS was evaluated in methanol as solvent and DPBF as the singlet oxygen sensor;

[0053] Figure 6 The superoxide anion generating ability of the target compound FNBS and the comparative molecules was evaluated in water using DHE as a superoxide anion sensor and mtDNA as a signal amplifier;

[0054] Figure 7 This is an experiment to combine the target compound FNBS with RNA extracted from cells after illumination;

[0055] Figure 8 The photosensitizer FNBS uptake experiment in mouse breast cancer (a) and live-dead staining experiment (b);

[0056] Figure 9 It is the evaluation of the killing ability of the target molecule FNBS on different tumor cells;

[0057] Figure 10 It is the detection of cell pyroptosis markers, (a) cell morphology changes, (b) ATP concentration detection, (c) LDH concentration detection:

[0058] Figure 11 It is the immunofluorescence staining of the cell immunogenic death-related factor HMGB-1;

[0059] Figure 12 The application of FNBS in in vivo imaging:

[0060] Figure 13 Figure 3: FNBS evaluation of the responsiveness of living immune cells, (a) changes in the content of dendritic cells within the tumor, and (b) the content of T cells in situ and distal tumors. DETAILED DESCRIPTION

[0061] Example 1

[0062] A photoactivated RNA anchoring photosensitizer, whose absorption and emission are in the infrared region, has the following structural formula:

[0063]

[0064] The preparation method of the phenothiazine photosensitizer is as follows:

[0065]

[0066] The specific preparation steps are as follows:

[0067] 1) Synthesis of Intermediate 1

[0068] N,N-diethyl-p-phenylenediamine (1 g, 6.09 mmol) was added to a stirred solution of aluminum sulfate (4.11 g, 6.52 mmol) in water (10 mL). Sodium thiosulfate (2.21 g, 14 mmol) and zinc chloride (0.872 g, 6.39 mmol) were added sequentially. The reaction mixture was cooled in an ice bath, and 4 mL of an aqueous solution of potassium dichromate (0.49 g, 1.68 mmol) was slowly added. The mixture was stirred in an ice bath for 2 h. The observed precipitate was filtered and washed with acetone. The crude product was refluxed in methanol (12 mL) and filtered to afford a dark gray solid (0.75 g) in a 45% yield. This product was used without further purification or characterization.

[0069] 2) Synthesis of Intermediate 2

[0070] 1-Amino-5,6,7,8-tetrahydronaphthylamine (147 mg, 1.0 mmol), 3-bromopropionic acid (182 mg, 1.5 mmol) and potassium carbonate (129.6 mg, 1.2 mmol) were dissolved in acetonitrile and refluxed. The reaction was monitored by TLC. After completion, the solvent was distilled off under reduced pressure and separated by column chromatography (dichloromethane:methanol, 100 / 1-20 / 1) to give a light yellow oily intermediate 2 (131.4 mg, yield: 60%). 1 HNMR(400MHz,MeOD)δ6.94(t,J=7.8Hz,1H),6.47(d,J=8.0Hz,1H),6.43(d,J=7.5Hz,1H),3.41(t,J=6.7Hz,2 H),2.69(t,J=6.2Hz,2H),2.61(t,J=6.7Hz,2H),2.40(t,J=6.4Hz,2H),1.88–1.81(m,2H),1.76–1.69(m,2H).

[0071] 3) Synthesis of Intermediate 3

[0072] Intermediate 2 (113.0 mg, 0.52 mmol), 1-hydroxybenzotriazole (83.5 mg, 0.62 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (95.9 mg, 0.62 mmol), and triethylamine (156.5 mg, 1.5 mmol) were dissolved in dichloromethane and stirred at room temperature for half an hour. Furan-2-methylamine (50.0 mg, 0.52 mmol) was then added and allowed to react with stirring at room temperature. The reaction was monitored by TLC. After completion of the reaction, water was added to extract the organic phase, and the solvent was removed by distillation under reduced pressure. The organic phase was separated by column chromatography (dichloromethane:methanol, 100 / 1 to 40 / 1) to obtain Intermediate 3 (77.5 mg, yield: 50%). 1 HNMR (400MHz, CDCl3) δ7.37 (s, 1H), 7.34 (s, 1H), 7.03 (t, J = 7.7Hz, 1H), 6. 54(d,J=7.5Hz,1H),6.50(d,J=7.9Hz,1H),6.33(s,1H),5.88(s,1H),4.27( d,J=5.4Hz,2H),3.51(t,J=5.8Hz,2H),2.73(t,J=5.7Hz,2H),2.54(t,J=5 .8Hz,2H),2.37(t,J=6.1Hz,2H),1.83(d,J=5.6Hz,2H),1.79–1.67(m,2H).

[0073] 4) Synthesis of the target compound FNBS

[0074] Intermediate 1 (158 mg, 0.57 mmol) and intermediate 3 (298 mg, 0.28 mmol) were dissolved in methanol and refluxed, followed by the addition of silver carbonate (276 mg, 0.57 mmol) in portions. The reaction was terminated by TLC tracking until the starting material disappeared, and the mixture was cooled to room temperature. The solid was removed by filtration, and the solvent was removed by distillation under reduced pressure. The title compound (70 mg, yield: 49%) was obtained by column chromatography (dichloromethane:methanol, 100 / 1-10 / 1). 1 H NMR(400MHz,MeOD)δ7.98(s,1H),7.45(d,J=2.7Hz,1H),7.37(s,1H),7.34(s,1H),7.32(s,1H),7.22(d,J=7.3Hz,1H),6.30(s,1H),4.19(s,2H),3 .82(d,J=6.6Hz,2H),3.79–3.71(m,4H),3.21(t,J=5.8Hz,2H),2.63(t,J =6.6Hz,2H),2.50(t,J=6.0Hz,2H),2.00–1.85(m,4H),1.38–1.34(m,5H).13 C NMR (101 MHz, MeOD) δ 172.96, 155.16, 153.65, 145.96, 144.49, 141.37, 139.67, 136.37, 135.50, 135.32, 129.96, 123.73, 119.41, 111.21, 106.24, 103.31, 55.11, 47.08, 41.28, 35.99, 35.08, 26.59, 25.05, 22.94, 22.68, 13.03. [M+H], calculated: 489.2318. Found: 489.2270.

[0075] Example 2

[0076] This example investigates the spectral changes of the photosensitizer (FNBS) prepared in Example 1 in different solvents and the types of reactive oxygen species generated after illumination.

[0077] (1) Determination of optical properties of prepared photosensitizers

[0078] FNBS solid was dissolved in DMSO to prepare a 5 mM stock solution and stored in the dark. The stock solution was diluted into various solvents (dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, ethyl acetate, phosphate buffer, acetone, toluene, and methanol) to a final photosensitizer concentration of 5 μM. Spectral data were recorded using UV and fluorescence spectrometers.

[0079] (2) Types of reactive oxygen species generated by photosensitizer (FNBS) after light exposure

[0080] The singlet oxygen generation capacity of FNBS was determined using the singlet oxygen scavenging agent 1,3-diphenylisobenzofuran (DPBF) and methylene blue as a reference. Dihydroethidium (DHE) was used as a superoxide anion scavenging agent (excitation wavelength: 561 nm, emission wavelength: 575-620 nm), and hydroxyphenylfluorescein (HPF) was used as a hydroxyl radical scavenging agent (excitation wavelength: 488 nm, emission wavelength: 500-540 nm). The concentration of the photosensitizer was 5 μM, and the concentration of the reactive oxygen species scavenger was 5 μM. Under a 660 nm LED light of 2 mW / cm 2 Under irradiation, the spectral changes at different time points were measured.

[0081] Experimental results: Figure 4 As shown in (left), the characteristic absorption peak of the photosensitizer FNBS is around 640nm and the wavelength red-shifts as the polarity decreases. Figure 4 (Right) It can be found that the characteristic emission peak of the photosensitizer is located in the near infrared region at 660nm. Figure 5As shown in the figure, the maximum absorption peak intensity of the DPBF illumination group alone did not change significantly, while the absorption of DPBF at 411nm in the photosensitizer plus illumination group gradually weakened with the extension of illumination time, and the decay rate of FNBS was faster than that of NBS, indicating that FNBS has a stronger ability to produce singlet oxygen than NBS. At the same time, the singlet oxygen yield of FNBS was calculated to be 0.32 by reference. The detection results of superoxide anions are shown in the figure. Figure 6 As shown, the fluorescence signal in the illumination-only group showed no significant change, while the fluorescence signal of DHE gradually increased in the photosensitizer plus illumination group. In summary, the reactive oxygen species produced by FNBS include singlet oxygen and superoxide anions. The singlet oxygen yield of FNBS, as high as 0.32, exceeds that of most reported heavy atom-free photosensitizers. Furthermore, the superoxide anions it produces can overcome the hypoxic environment of tumors, maintaining high photodynamic therapy efficacy even in hypoxic environments.

[0082] Example 3

[0083] This example studies the RNA binding ability of the photosensitizer prepared in Example 1, its uptake ability by tumor cells, its reactive oxygen species generation ability, and its cytotoxicity.

[0084] (1) Binding ability of photosensitizer FNBS to RNA

[0085] Tumor cells in the logarithmic growth phase were lysed and RNA was extracted. The cells were divided into five groups and set up six experimental groups, namely blank group, photosensitizer concentration gradient group (1, 2, 3, 4, 5 μM). After mixing the photosensitizer with RNA, the cells were illuminated by a 660 nm LED light (2.0 mW / cm 2 After irradiation (10 min), the cells were incubated for half an hour. After centrifugation, the supernatant was removed and added to a nucleic acid gel for electrophoresis using a Tris-Acetate-EDTA system at 150 V. Gel electrophoresis imaging was performed when the cells reached the middle of the run. The excitation wavelength of the nucleic acid dye EB was 480 nm, and the emission wavelength was 520 nm. The excitation wavelength of the photosensitizer FNBS was 640 nm, and the emission wavelength was 660 nm.

[0086] (2) Tumor cell uptake of the photosensitizer FNBS

[0087] In this example, mouse breast cancer cells were selected as the experimental target, and 1*10 5 Cells were seeded in a confocal dish and cultured in a cell culture incubator until the cells reached the logarithmic growth phase. The culture medium was then discarded and the cells were washed 2-3 times with PBS. The photosensitizer FNBS was diluted to 1 μM in the culture medium and added to the dish. The fluorescence signal was observed under a fluorescence confocal microscope (excitation wavelength: 640 nm, emission: 655-700 nm).

[0088] (3) Cytotoxicity and live-dead staining experiments: including phototoxicity and dark toxicity experiments. The specific procedures are as follows:

[0089] Phototoxicity experiment group: Four different tumor cells were seeded into 96-well plates at a density of 10,000 cells per well and incubated in an incubator for 24 hours. Cells with poor adhesion were washed away with PBS. Different concentrations of photosensitizer FNBS (0, 20, 40, 80, 160, 320, 640 nM) were added to each well, with 5 replicates for each concentration. The cells were incubated for another half hour and then illuminated (660 nm, 2 mW / cm 2 , 10 min). After 24 hours, the culture medium was discarded and 200 μL of 0.5 mg / mL MTT was added to each well. Four hours later, the liquid in the 96-well plate was discarded and 100 μL of dimethyl sulfoxide was added. The absorbance at 490 nm and 570 nm was measured using a microplate reader, and the cell viability was calculated.

[0090] Dark toxicity experimental group: The experimental steps are as follows, but this group does not require light. After adding photosensitizer and incubating, the cells are cultured for another 24 hours before adding MTT. After 4 hours, dimethyl sulfoxide is added. The absorbance at 490 nm and 570 nm is measured using a microplate reader, and the cell viability is calculated.

[0091] Live dead staining: The experimental steps are as follows; 1*10 5 Cells were seeded in a confocal dish and cultured in a cell culture incubator until the cells reached the logarithmic growth phase. The culture medium was then discarded and the cells were washed 2-3 times with PBS. The cells were then divided into four groups: a control group, a control group plus illumination group, a photosensitizer group, and a photosensitizer plus illumination group. AM / PI was added to each of the four groups. Simultaneously, the photosensitizer FNBS was diluted to 1 μM in culture medium and added to the photosensitizer group and the photosensitizer plus illumination group (illumination was applied 0.5 h after addition). After incubation, the cells were observed for fluorescence signals under a fluorescence confocal microscope. AM excitation wavelength was 488 nm, with an acceptance range of 500-540 nm. PI excitation wavelength was 535 nm, with an acceptance range of 550-620 nm.

[0092] Experimental results: The results are as follows Figure 7 As shown, the RNA bands in the control group are clear, and the bands where the nucleic acid dye EB is located are clear, indicating that the RNA extraction from the cells is correct. After exposure to different concentrations of photosensitizer and illumination, red fluorescent signal bands appeared under 640nm excitation, and they completely overlapped with the RNA signal, indicating that FNBS can bind to RNA under illumination to achieve the purpose of photoactivation labeling, and also lay the foundation for the subsequent generation of reactive oxygen species under illumination to damage RNA. Figure 8 As shown in Figure a, the intracellular fluorescence signal intensity reaches a plateau 10 minutes after addition, and there is almost no difference in the intensity at 15 minutes compared with that at 10 minutes, indicating that FNBS can be quickly taken up by tumor cells. Figure 8As shown in Figure b, the control group, the control plus light group, and the photosensitizer-only group all showed bright green fluorescence signals, indicating that the light dose or photosensitizer concentration did not cause damage to tumor cells, while the photosensitizer plus light group showed bright red signals, indicating that the photosensitizer had excellent tumor cell killing ability. Figure 9 As shown, mouse breast cancer cells (4T1), human breast cancer cells (MD-MB-231), and human liver cancer cells (HepG2) were used as experimental subjects. FNBS exhibited negligible dark toxicity, demonstrating its good in vitro biocompatibility. However, after illumination, increasing the concentration of the photosensitizer FNBS decreased cell survival. To visualize the cell-killing ability of FNBS, a live-death assay and fluorescence confocal microscopy were used.

[0093] From the above experimental results, it can be seen that FNBS can produce reactive oxygen species and induce cell death under extremely low light doses.

[0094] Example 4

[0095] This example studies the use of photosensitizer FNBS to label RNA to induce cell pyroptosis and immunogenic cell death and evaluate in vivo immune responses.

[0096] Based on previous cytotoxicity experiments, we speculated that FNBS binding to RNA may induce cellular metabolic disorders and ultimately lead to cell immunogenic death. Therefore, we further studied the detection of markers related to FNBS-mediated cell immunogenic death, as follows:

[0097] (1) Detection of cell pyroptosis and its markers

[0098] 10,000 cells were seeded in a confocal microplate dish and allowed to fully adhere to the wall and grow to a density of 80%. After washing with PBS 2-3 times, the photosensitizer FNBS (1 μM) diluted in PBS was added. After incubation at room temperature for half an hour, the cells were illuminated and their morphology was observed by confocal microscopy. The ATP and lactate dehydrogenase (LDH) assay kits were then used to measure changes in the levels of the corresponding targets.

[0099] (2) Detection of cell death markers of immunogenicity

[0100] Immunofluorescence staining was used to detect the cell death marker high mobility group protein (HMGB-1). Ten thousand cells were seeded in a confocal dish and divided into four groups: control group, control plus light group, FNBS group, and FNBS plus light group. After the cell density reached 80%, PBS was washed 2-3 times. The latter two groups were added with the photosensitizer FNBS (1 μM) diluted in PBS. After incubation at room temperature for half an hour, the FNBS plus light group was illuminated (660 nm, 2 mW / cm 2, 10min). Then, the culture medium of the four groups was poured out and washed 2-3 times with PBS. 1mL of 4% paraformaldehyde was added to each confocal dish and fixed at 4°C for 20min. After pouring out the fixative, add PBS and shake on a flatbed shaker. Wash three times, 5min each time. After the end, add 1% Trition-100 for permeabilization, pour it out after half an hour and wash three times with PBS. Add blocking solution and block for half an hour and wash three times with PBS. Then add primary antibody (CRT or HMGB-1, diluted 1000 times) and incubate overnight. The next day, remove the primary antibody dilution solution and wash three times with PBS. Then add secondary antibody containing fluorescent label (diluted 1000 times), incubate at room temperature for two hours, wash three times with PBS, add 0.5μL DAPI dye (dissolved in DMSO, 5mM) and observe the fluorescence signal by fluorescence confocal microscopy.

[0101] (3) In vivo imaging and immunoassay

[0102] Twenty Balb-C female mice (5-6 weeks old) were subcutaneously injected with 1*10 6 Mouse breast cancer cells, when the tumor volume is about 100mm 3 Then, the mice were randomly divided into four groups, including a control group (only 100 μL of saline solution was injected), a control plus light group (100 μL of saline solution was injected, and 660 nm LED light 50 mW / cm 2 , 10 min), photosensitizer (only injection of photosensitizer 10 nM), photosensitizer plus light (photosensitizer 10 nM and 660 nm LED light 50 mW / cm 2 , 10 minutes). The photosensitizer was first injected through the tail vein to observe the accumulation time of the photosensitizer in the living tumor to determine the optimal treatment time. Photodynamic therapy was then performed based on the determined optimal tumor accumulation time. On the seventh day of treatment, tumors in each experimental group were dissected and analyzed for dendritic cell and T cell expression.

[0103] Experimental results: Figure 10 As shown in Figure a, when the photosensitizer FNBS was added and the cells were illuminated, vesicles (blue arrows) were observed around the cell membrane at 5 minutes, and the number of vesicles increased with time, indicating that pyroptosis may occur under illumination. The ATP content and extracellular LDH in the cells were then measured. Figure 10 As shown in Figure b, the ATP content in the cells gradually decreases with the increase of photosensitizer concentration. Figure 10 As shown in Figure c, the extracellular concentration of LDH increases with the concentration of photosensitizer. In summary, FNBS can induce cell pyroptosis under light. Figure 11As shown in the figure, HMGB-1, a marker of cell immunogenic death, was measured. There was no significant change in the HMGB-1 signal in the nuclei of the control, light-only, and FNBS groups. However, the fluorescence signal in the cells of the photosensitizer FNBS plus light group was significantly weakened, indicating the migration of HMGB-1. No fluorescence signal was observed in the other groups. Figure 12 As shown in the figure, after the photosensitizer is injected into the tail vein, the accumulation of the photosensitizer in the tumor increases with time, reaching a maximum at 25 minutes, then gradually decaying, and almost no fluorescence after 3 hours. This shows that the photosensitizer molecule can be quickly enriched in tumor cells and metabolized quickly in the body, which has better biosafety. Figure 13 As shown,

[0104] The above results indicate that FNBS can cause cell pyroptosis under light, thereby inducing immunogenic cell death, enhancing the body's immune response and achieving photoimmunotherapy.

[0105] In summary, FNBS, as a new photosensitizer, not only has excellent photochemical properties and reactive oxygen species generation capacity, but also exhibits targeting, cytotoxicity, and immunomodulatory effects in tumor treatment. It has the potential to be developed into a highly effective photodynamic therapy drug. Its properties include:

[0106] 1. Optical properties and active oxygen generation ability:

[0107] FNBS exhibits stable optical properties in different solvents, with a characteristic absorption peak around 640 nm, which red-shifts with changes in solvent polarity, indicating that it has good solubility and photostability.

[0108] Under light conditions, FNBS can efficiently generate singlet oxygen ( 1 O2), with a yield as high as 0.32, which is better than most known heavy atom-free photosensitizers, demonstrating its excellent photochemical performance.

[0109] FNBS can also generate superoxide anions, a property that enables it to maintain efficient photodynamic therapy (PDT) effects in the hypoxic environment of tumors.

[0110] 2. RNA binding ability and tumor cell uptake:

[0111] FNBS can effectively bind to cytoplasmic RNA and achieve photoactivation labeling under light conditions, which may kill tumor cells by destroying RNA.

[0112] Tumor cells can quickly take up FNBS, which is conducive to its accumulation at the tumor site and improves the targeted treatment.

[0113] 3. Cytotoxicity and phototoxicity:

[0114] FNBS showed low cytotoxicity under dark conditions, indicating its good in vitro biocompatibility.

[0115] Under light, FNBS showed obvious cytotoxicity. The higher the concentration, the lower the cell survival rate, indicating that its photodynamic effect is significant.

[0116] 4. Cell pyroptosis and immunogenic death:

[0117] FNBS can induce cell pyroptosis, which is manifested by the formation of cell membrane vesicles, decreased ATP levels, and increased LDH release.

[0118] FNBS can also promote cell immunogenic death, such as the release of HMGB-1, which may activate the body's immune system and have a long-term inhibitory effect on tumors.

[0119] 5. In vivo application and immune response:

[0120] FNBS was rapidly accumulated in tumor tissue after tail vein injection, indicating that it has good in vivo distribution characteristics.

[0121] After treatment, FNBS can increase the expression of dendritic cells and T cells in the tumor site, activate the body's immune response, and achieve photoimmunotherapy effects.

[0122] It should be understood that the above embodiments are intended only to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection thereof. Persons skilled in the art may make various modifications and variations to the above embodiments without departing from the spirit and basic principles of the present invention, but such modifications and variations shall still fall within the scope of protection of the claims of the present invention and their equivalents. The scope of protection of the present invention shall be based on the appended claims and shall not be limited to the details of the above embodiments.

Claims

1. A photoactivated RNA-labeled phenothiazine photosensitizer having the structure shown in Formula I below: The R1 is selected from one of dimethylamino, diethylamino, aziridine, azetidine, aziridine and aziridine.

2. The method for preparing a photosensitizer according to claim 1, wherein: The following steps are involved: 1) In an aqueous solution of aluminum sulfate, an aniline derivative is mixed with sodium thiosulfate and zinc chloride, and an aqueous solution of potassium dichromate is added. The mixture is stirred at low temperature, and the precipitate is filtered and washed. The crude product is refluxed in methanol and filtered to obtain intermediate 2; 2) 1-amino-5,6,7,8-tetrahydronaphthylamine was refluxed with 3-bromopropionic acid and potassium carbonate in acetonitrile, and the intermediate 3 was obtained by column chromatography; 3) Intermediate 2 was stirred with 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and triethylamine in dichloromethane at room temperature, furan-2-methylamine was added, and intermediate 4 was obtained by column chromatography. 4) Intermediate 2 and intermediate 4 were refluxed in methanol under the catalysis of silver carbonate, and the target compound FNBS was separated by column chromatography.

3. The method according to claim 2, wherein: In the step 1), the molar ratio of 1-amino-5,6,7,8-tetralin, 3-bromopropionic acid and potassium carbonate is (0.8-1.2):(1-2):(1-2).

4. The method according to claim 2, wherein: In the step 1), the aniline derivative is selected from one of N,N-dimethyl-p-phenylenediamine, 4-aziridine-based aniline, 4-azetidinyl aniline, 4-azacyclopentyl aniline and 4-azacyclohexyl aniline.

5. The method according to claim 2, wherein: In the step 1), the molar ratio of the aniline derivative, aluminum sulfate, sodium thiosulfate, zinc chloride, and potassium dichromate is 1:(1-2):(1-2):(0.1-0.5).

6. The method according to claim 2, wherein: In the step 2), the molar ratio of 1-amino-5,6,7,8-tetrahydronaphthylamine, 3-bromopropionic acid, and potassium carbonate is 1:(1-2):(1-2); The molar ratio of the intermediate 1, 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, triethylamine and furan-2-methylamine in step 3) is 1:(1-2):(1-2):(1-2):(1-3); The molar ratio of intermediate 2 to intermediate 4 in step 4) is 1:1-3.

7. Use of the photosensitizer according to claim 1 in the preparation of drugs for non-disease diagnosis and treatment.

8. The application according to claim 7, characterized in that: The application relates to the field of inhibiting distal tumor growth and achieving photoimmunotherapy effects. The photosensitizer promotes immune response and cell-selective effects through its photodynamic properties.

9. The use according to claim 7, characterized in that: It includes the preparation of drugs for photodynamic therapy; the preparation of reagents for RNA labeling and research; the preparation of drugs for tumor immunotherapy; and the preparation of reagents for cell pyroptosis research.

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