Targeted photosensitizer based on phenothiazine cell membrane as well as preparation method and application of targeted photosensitizer

Abstract

The invention discloses a phenothiazine-based cell membrane targeting photosensitizer and a preparation method and application thereof, the structure of the photosensitizer is as shown in formula I. The photosensitizer is based on phenothiazine photosensitizer, and MNBS is obtained through non-covalent connection of a long alkane chain and a sulfonate radical structure. The photosensitizer can be rapidly taken by tumor cells and targeted to cell membranes, and meanwhile, the photosensitizer generates active oxygen under illumination to cause damage to the cell membranes and induce pyroptosis of the cells, so that photoimmunotherapy is realized. The photosensitizer has a relatively strong killing capability on tumor cells, and the anti-tumor effect of the photosensitizer comprises two parts: the killing effect of active oxygen generated by the photosensitive dye on the tumor cells; the pyroptosis of the cells is induced, and the cell immunogenicity death is caused. The photosensitizer disclosed by the invention has the advantages that the photosensitizer can cause pyroptosis while performing photodynamic therapy on cells under an extremely low light dose, so that photoimmunotherapy is realized, and the anti-tumor curative effect is improved.

Description

Technical Field

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

[0002] The cell membrane is mainly composed of a phospholipid bilayer, glycolipids, and glycoproteins, and is an important barrier for cells. The cell membrane regulates and selects the entry and exit of substances while maintaining the homeostatic environment within the cell. It mainly absorbs, digests, and excretes substances inside and outside the cell membrane through endocytosis, phagocytosis, or exocytosis, and is essential for cell physiological activities. In addition, part of the reason for drug resistance during long-term drug treatment is the poor efficacy caused by the improvement of cell membrane-related efflux function. Therefore, the cell membrane can be used as a potential target for treatment to destroy the homeostasis of diseased cells to achieve the purpose of treatment.

[0003] Photodynamic therapy is a new type of non-invasive treatment method that applies the principles of photochemistry, photophysics and photobiology to the diagnosis and treatment of diseases. However, existing photosensitizers have significant limitations in clinical applications: First, most photosensitizers (such as cyanines, phthalocyanines and phenothiazines) perform poorly in terms of photostability, biocompatibility and photosensitization efficiency. For example, cyanine photosensitizers have poor photostability, phthalocyanines and fluoroboron dipyrrole photosensitizers have poor aggregation quenching and biocompatibility in aqueous solutions due to their large conjugated planar structures, and phenothiazine photosensitizers (such as methylene blue) have problems such as low photosensitization efficiency, high dark toxicity and short triplet lifetime. Secondly, existing photosensitizers usually rely on the type II photodynamic mechanism (generating singlet oxygen through energy transfer), and their efficacy is significantly reduced in the hypoxic tumor microenvironment. In addition, existing photosensitizers are difficult to achieve efficient treatment at low light doses, and lack the ability to induce immunogenic death, and cannot effectively inhibit tumor metastasis and recurrence.

[0004] Therefore, developing a photosensitizer that can efficiently generate ROS at low light doses, has cell membrane targeting capabilities, and maintains efficient therapeutic effects under hypoxic conditions has become a key issue that needs to be urgently addressed in the field of photodynamic therapy. Summary of the invention

[0005] The present invention aims to solve the problems of insufficient efficacy, poor targeting and limited immune activation ability of existing photosensitizers in hypoxic environments, and provides a design and application of phenothiazine-based photosensitizers. Through innovative phenothiazine dye design and cell membrane targeting mechanism, the present invention achieves low light dose (3J / cm 2 ) under high-efficiency photodynamic therapy and immune activation, significantly improving the effect of photodynamic therapy while reducing side effects. The present invention includes the structural design of photosensitizers, preparation methods and their applications in photodynamic therapy, tumor immunotherapy and cell pyroptosis research, providing a new strategy for tumor treatment.

[0006] In the first aspect of the present invention, a phenothiazine cell membrane-targeted photosensitizer is provided, and the photosensitizer has the structure shown in the following formula I:

[0007]

[0008] The R selected from nitrogen-containing functional groups; Further preferably, the nitrogen-containing functional group is selected from one of dimethylamino, diethylamino, aziridine, azetidine, azolidine, cyclohexylamine.

[0009] Further preferably, the photosensitizer is selected from one of C1, C2, C3, C4, C5, C6;

[0010]

[0011] The compound has the following characteristics;

[0012] The main absorption peak is located at about 640 nm ± 20 nm, and the characteristic emission peak is located at 660 nm ± 20 nm in the near-infrared region;

[0013] Under light illumination conditions, the singlet oxygen yield is as high as 0.28 ± 0.05;

[0014] Quickly taken up by target cells, and basically completed within 15 minutes, and located on the cell membrane of tumor cells;

[0015] The cell survival rate in the group with only the photosensitizer added is higher than 90%, indicating that there is no obvious toxicity to cells under dark conditions; Under normoxic conditions, when irradiated with a very low light dose (3 mW / cm 2 , 10 min), the cell survival rate drops to 20% ± 5%, and at the same time triggers pyroptosis to induce immunogenic death and improve the body's immune response.

[0016] The photosensitive dye molecule MNBS is connected by a substitution reaction of a phenothiazine-based photosensitive dye platform and a long alkane chain structure.

[0017] In the second aspect of the present invention, a preparation method of the photosensitizer described in the first aspect is provided, including the following steps:

[0018]

[0019] The specific preparation steps are as follows:

[0020] 1) In an aqueous solution of aluminum sulfate, an aniline derivative is mixed with sodium thiosulfate and zinc chloride, an aqueous solution of potassium dichromate is added, stirred at low temperature, filtered, and the precipitate is washed with organic solvent I. The crude product is refluxed in methanol and filtered to obtain intermediate 1;

[0021] 2) 1-Amino-5,6,7,8-tetrahydronaphthylamine is refluxed with 1-bromo-3-chloropropane and potassium carbonate in organic solvent II, and the intermediate 2 is purified and separated;

[0022] 3) Intermediate 2 is refluxed with N-methyl-1-dodecylamine, potassium iodide and potassium carbonate in organic solvent III, and the intermediate 3 is purified and separated;

[0023] 4) Intermediate 3 is refluxed with 1,3-propane sultone in organic solvent IV, and the intermediate 4 is purified and separated;

[0024] 5) Intermediate 4 is refluxed with intermediate 1 and silver carbonate in organic solvent V, and the target product MNBS is purified and separated.

[0025] For the technical solution described above, more preferably, the aniline derivative in step 1) is preferably N,N-dimethyl-p-phenylenediamine, 4-aziridinyl aniline, 4-azetidinyl aniline, 4-azacyclopentyl aniline, 4-azacyclohexyl aniline, etc.; more preferably N,N-diethyl-p-phenylenediamine.

[0026] For the technical solution described above, more preferably, the molar ratio of the aniline derivative, aluminum sulfate, sodium thiosulfate, zinc chloride, and potassium dichromate in step 1) is 1:1-2:2-3:1-2:0.1-0.5; more preferably the molar ratio is 1:1-1.5:2-2.5:1-1.5:0.2-0.3.

[0027] For the technical solution described above, the concentration range of the potassium dichromate aqueous solution in step 1) is 0.1-1 mM; more preferably the concentration range is 0.3-0.5 mM.

[0028] For the technical solution described above, more preferably, the low temperature in step 1) is 0-5 °C; more preferably in an ice-water bath.

[0029] For the technical solution described above, more preferably, the organic solvent I in step 1) is one of acetone, pyridine or dioxane.

[0030] For the technical solution described above, more preferably, the molar ratio of 1-amino-5,6,7,8-tetrahydronaphthalene, 1-bromo-3-chloropropane and potassium carbonate in step 2) is 0.8-1.2:1-2:1-2; more preferably the molar ratio is 1:1.5-2:1.5-2.

[0031] For the technical solution described above, more preferably, the organic solvent II in step 2) is one of ethanol, acetonitrile, dichloromethane or dimethyl sulfoxide.

[0032] For the technical solution described above, more preferably, the purification and separation in step 2) is column chromatography, and the eluent for separation is a mixture of petroleum ether and dichloromethane in a volume ratio of 20 - 100:1.

[0033] For the technical solution described above, more preferably, the molar ratio of intermediate 2, N - methyl - 1 - dodecylamine, potassium iodide, and potassium carbonate in step 3) is 1:1 - 2:1 - 2:1 - 2. More preferably, the molar ratio is 1:1.5 - 2:1.5 - 2:1.5 - 2, and the most preferred molar ratio is 1:2:2:2.

[0034] For the technical solution described above, more preferably, the organic solvent III in step 3) is one of acetonitrile, ethanol, petroleum ether, or dimethyl sulfoxide.

[0035] For the technical solution described above, more preferably, the purification and separation in step 3) is column chromatography, and the separation eluent is a mixture of methanol and dichloromethane with a volume ratio of 1:10 - 100.

[0036] For the technical solution described above, more preferably, the molar ratio of intermediate 3 and 1,3 - propane sultone in step 4) is 1:1 - 2, and the most preferred is 1:1.

[0037] For the technical solution described above, more preferably, the purification and separation in step 4) is column chromatography, and the separation eluent is a mixture of methanol and dichloromethane with a volume ratio of 1:5 - 100.

[0038] For the technical solution described above, more preferably, the organic solvent IV in step 4) is one of acetonitrile, ethanol, or dichloromethane.

[0039] For the technical solution described above, more preferably, the molar ratio of intermediate 4, intermediate 1, and silver carbonate in step 5) is 1:1 - 3:1 - 2. More preferably, the molar ratio is 1:1.5 - 2.5:1.5 - 2, and the most preferred is 1:2:2.

[0040] For the technical solution described above, more preferably, the purification and separation in step 5) is column chromatography, and the separation eluent is a mixture of methanol and dichloromethane with a volume ratio of 1:10 - 100.

[0041] For the technical solution described above, more preferably, the organic solvent IV in step 5) is one of methanol, ethanol, ethylene glycol, or dimethyl sulfoxide.

[0042] The third aspect of the present invention is to protect the application of the compound in the preparation of preparations for non-disease diagnosis and treatment, including the preparation of preparations for photodynamic therapy, tumor immunotherapy and pyroptosis research, wherein the photosensitizer promotes immune response and cell-selective action through its unique photodynamic properties.

[0043] Specifically, the application directions of the photosensitizer include the preparation of photodynamic therapy, tumor immunotherapy and pyroptosis preparations for non-disease diagnosis and treatment:

[0044] Preparing a drug for photodynamic therapy; activating the production of reactive oxygen species by light irradiation to achieve precise killing of target cells; the killing efficiency of target cells reaches 90% ± 5% at extremely low drug doses and light doses (1 μM, 3 mW / cm 2 Light irradiation for 10 min);

[0045] Preparing a drug for tumor immunotherapy, enhancing the immune response of the body to target cells by inducing immunogenic death; the dendritic cell immune response is increased by about 12 times, and the T cell immune response efficiency is increased by about 2 times.

[0046] Preparing a reagent for pyroptosis research, as a research tool, to explore the mechanism of pyroptosis and understand its role in the disease process.

[0047] More preferably, the application of the compound in the preparation of drugs for photodynamic therapy and / or immunotherapy, the photosensitizer can target the tumor cell membrane, generate type I and type II reactive oxygen species under light irradiation, and promote the anti-tumor effect. More specifically, the compound specifically binds to the cell membrane of tumor cells, realizes precise labeling and efficient destruction of tumor cells, and at the same time enhances the immune response of the body to tumors by activating T lymphocytes, significantly reducing the risk of tumor metastasis and recurrence.

[0048] Advantages and beneficial effects of the present invention:

[0049] 1. Optical properties and reactive oxygen species generation ability

[0050] Compared with the prior art, the main absorption peak of MNBS is located at about 640 nm ± 20 nm, and the characteristic emission peak is located at 660 nm ± 20 nm, having better near-infrared absorption and emission characteristics, suitable for deep tissue treatment.

[0051] Compared with traditional photosensitizers, the singlet oxygen yield of MNBS under light irradiation is as high as 0.28 ± 0.05, which is significantly better than most non-heavy atom photosensitizers.

[0052] MNBS also generates superoxide anions through type I photodynamic mechanism, significantly reducing the dependence of photodynamic therapy on oxygen and maintaining high-efficiency photodynamic therapy under hypoxic conditions, while the efficacy of traditional type II photosensitizers (such as phthalocyanines) significantly decreases in hypoxic environments.

[0053] 2. Cellular uptake and targeting ability

[0054] Compared with the prior art, MNBS can be rapidly taken up by target cells, and the uptake is basically completed within 15 minutes, and specifically localizes to the cell membrane of tumor cells, significantly improving the accuracy of treatment. Traditional photosensitizers (such as cyanines) usually require longer uptake time and lack the ability of cell membrane targeting.

[0055] 3. Cytotoxicity and phototoxicity

[0056] Compared with existing photosensitizers, the cell viability of MNBS is higher than 90% under dark conditions, indicating that its dark toxicity is extremely low, which is better than many traditional photosensitizers (such as phenothiazine photosensitizers usually have relatively high dark toxicity).

[0057] Under normoxic conditions, after MNBS is irradiated with a very low light dose (3 mW / cm 2 , 10 min), the cell viability drops to 20% ± 5%, which is significantly better than the treatment effect of existing photosensitizers under the same conditions.

[0058] 4. Pyroptosis and immune activation ability

[0059] Compared with the prior art, MNBS induces pyroptosis under light irradiation, manifested as the formation of cell membrane vesicles, a decrease in ATP content and an increase in LDH release, while traditional photosensitizers usually only induce apoptosis and cannot effectively activate the immune response.

[0060] MNBS significantly improves the immune response of the body by inducing immunogenic cell death, such as the migration of HMGB1 and the activation of dendritic cells and T cells, providing a new strategy for photoimmunotherapy, while existing photosensitizers lack such immune activation ability.

[0061] 5. In vivo application and biosafety

[0062] Compared with traditional photosensitizers, MNBS rapidly accumulates in tumor tissues, reaches the peak within 4 hours, and is almost completely metabolized after 24 hours, showing better biodistribution and safety.

[0063] The low light dose requirement of MNBS (50 mW / cm 2 , 10 min) reduces the risk of collateral damage to healthy tissues, while existing photosensitizers usually require higher light doses to achieve similar effects.

[0064] 6. Wide application potential

[0065] Compared with the prior art, MNBS is not only applicable to the photodynamic therapy of solid tumors, but also can be used as a tool for studying the mechanism of pyroptosis, providing a new research platform for exploring the mechanism of cell death and immune regulation.

[0066] Its near-infrared absorption and emission characteristics endow it with potential application value in deep tissue imaging and therapy, while the effect of traditional photosensitizers in deep tissue therapy is limited. Description of the drawings

[0067] Figure 1 is the 1H NMR spectrum of the target compound MNBS;

[0068] Figure 2 is the 13C NMR spectrum of the target compound MNBS;

[0069] Figure 3 is the high-resolution mass spectrum of the target compound MNBS;

[0070] Figure 4 is the spectrum of the target compound MNBS in different solvents. (a) UV-Vis absorption spectrum, (b) fluorescence spectrum;

[0071] Figure 5 is the evaluation of the ability of the target compound MNBS to generate reactive oxygen species in water with DCFH as the reactive oxygen detection reagent;

[0072] Figure 6 is the evaluation of the ability of the target compound MNBS to generate singlet oxygen in methanol as the solvent and DPBF as the singlet oxygen sensor;

[0073] Figure 7 is the evaluation of the ability of the target compound MNBS to generate superoxide anions in water with DHE as the superoxide anion sensor and adding mtDNA as a signal amplifier;

[0074] Figure 8 is the uptake experiment (a) and live / dead staining experiment (b) of the photosensitizer MNBS in mouse breast cancer cells;

[0075] Figure 9 is the co-localization result of the target molecule MNBS with different commercial organelle dye probes;

[0076] Figure 10 is the evaluation of the killing ability of the target molecule MNBS on different tumor cells under normoxic and hypoxic conditions;

[0077] Figure 11 is the detection of pyroptosis markers (a) cell morphological changes, (b) ATP concentration detection, (c) LDH concentration detection:

[0078] Figure 12 is the immunofluorescence staining of the cell immunogenic death-related factor HMGB1;

[0079] Figure 13 is the application of MNBS in in vivo imaging:

[0080] Figure 14 is the evaluation of the response ability of MNBS to in vivo immune cells (a) the activation of dendritic cells in the spleen, (b) the changes in T cell activation in tumors. Specific Embodiments

[0081] The following will further describe in detail the specific embodiments of the present invention in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but not to limit the scope of the present invention.

[0082] In the specific implementation process of the present invention, in addition to the key steps described in detail, a variety of conventional chemical reagents and standard operation procedures are also used. For example, all solvents such as dichloromethane, N,N-dimethylformamide, methanol, etc. are purchased from commercial suppliers and their purity is ensured. In addition, common chemical reaction conditions such as TLC monitoring, rotary evaporation under reduced pressure, silica gel column chromatography, etc. are all standard operations in the field of organic synthesis.

[0083] Particularly, in the synthesis steps of the phenothiazine photosensitizer MNBS involved in the present invention, the preparation of intermediate 2 to intermediate 4 has undergone strict condition optimization to ensure the high purity and high yield of the target product MNBS. The optical properties, reactive oxygen species generation ability and cell uptake experiments of the photosensitizer are all verified by standardized spectroscopic and biological methods. The specific experimental conditions and methods are detailed in the example part.

[0084] The selection of these conventional steps and reagents is based on their wide applicability and reliability, and does not constitute a key impact on understanding the core technology of the present invention, so they are not described in detail.

[0085] Example 1

[0086] A cell membrane-targeted photosensitizer, whose absorption and emission are located in the infrared region, and the structural formula is as follows:

[0087]

[0088] The preparation method of the phenothiazine photosensitizer is as follows:

[0089]

[0090] The specific preparation steps are as follows:

[0091] 1) Synthesis of Intermediate 1

[0092] N,N - diethyl - p - phenylenediamine (1 g, 6.09 mmol) was added to a stirred aqueous solution (10 mL) of aluminum sulfate (4.11 g, 6.52 mmol). Sodium thiosulfate (2.21 g, 14 mmol) and zinc chloride (0.872 g, 6.39 mmol) were added successively. 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 added slowly. After stirring in the ice bath for 2 h, the observed precipitate was filtered and washed with acetone. The crude product was refluxed in methanol (12 mL), and a dark - gray solid (0.75 g) was obtained by filtration with a yield of 45%. This product can be used without further purification or characterization.

[0093] 2) Synthesis of Intermediate 2

[0094] 1 - Amino - 5,6,7,8 - tetrahydronaphthylamine (2 g, 13.6 mmol), 1 - bromo - 3 - chloropropane (4.24 g, 27.2 mmol) and potassium carbonate (3.76 g, 27.2 mmol) were dissolved in ethanol and refluxed. The reaction was monitored by TLC. After completion, the solvent was removed by distillation under reduced pressure, and the product was separated by column chromatography (petroleum ether:dichloromethane, 100 - 20:1) to obtain a pale - yellow oily Intermediate 2 (1.4 g, yield: 46%). 1 HNMR (400 MHz, Chloroform - d) δ7.05 (t, J = 7.8 Hz, 1H), 6.56 (d, J = 7.6 Hz, 2H), 3.66 (t, J = 6.2 Hz, 2H), 3.38 (t, J = 6.6 Hz, 2H), 2.75 (t, J = 6.2 Hz, 2H), 2.48–2.40 (m, 2H), 2.14 (p, J = 6.4 Hz, 2H), 1.92–1.82 (m, 2H), 1.80–1.71 (m, 2H).

[0095] 3) Synthesis of Intermediate 3

[0096] Intermediate 2 (1 g, 4.48 mmol), N - methyl - 1 - dodecylamine (1.78 g, 8.97 mmol), potassium iodide (1.49 g, 8.97 mmol) and potassium carbonate (1.24 g, 8.97 mmol) were dissolved in acetonitrile and refluxed. The reaction was monitored by TLC spotting. After completion, potassium carbonate and potassium iodide were removed by suction filtration, and the organic solvent was removed by distillation under reduced pressure. The residue was dissolved in 50 mL of dichloromethane, and the organic phase was extracted with saturated brine (3×50 mL). After drying over anhydrous sodium sulfate to remove water, the solvent was removed by distillation under reduced pressure, and the product was separated by column chromatography (dichloromethane:methanol, 100:10:1) to obtain a yellow oily Intermediate 3 (0.8 g, yield: 52%). 11H NMR (400 MHz, Chloroform-d) δ 7.02 (t, J = 7.8 Hz, 1H), 6.48 (d, J = 7.6 Hz, 1H), 6.42 (d, J = 8.0 Hz, 1H), 4.58 (s, 1H), 3.22 (q, J = 5.4 Hz, 2H), 2.73 (t, J = 6.1 Hz, 2H), 2.54 (s, 2H), 2.39 (t, J = 6.4 Hz, 4H), 2.29 (s, 3H), 1.86 (dtd, J = 8.9, 6.3, 2.4 Hz, 4H), 1.78–1.70 (m, 2H), 1.31–1.23 (m, 18H), 0.88 (t, J = 6.8 Hz, 3H).

[0097] 4) Synthesis of Intermediate 4

[0098] Dissolve Intermediate 3 (0.5 g, 1.29 mmol) and 1,3 - propane sultone (158 mg, 1.29 mmol) in acetonitrile and reflux. Monitor the reaction by TLC plate spotting. After the reaction is completed, remove the solvent by distillation under reduced pressure, and separate by column chromatography (dichloromethane:methanol, 100 - 5:1) to obtain white solid Intermediate 4 (0.3 g, yield: 48%). 1 1H NMR (400 MHz, Chloroform-d) δ 7.00 (t, J = 7.7 Hz, 1H), 6.56 (d, J = 7.7 Hz, 2H), 3.78–3.62 (m, 2H), 3.62–3.51 (m, 2H), 3.48 (s, 1H), 3.36 (d, J = 6.2 Hz, 2H), 3.12 (d, J = 11.8 Hz, 5H), 2.94 (d, J = 6.7 Hz, 2H), 2.71 (t, J = 6.2 Hz, 2H), 2.51 (t, J = 6.5 Hz, 2H), 2.25 (s, 2H), 2.16 (d, J = 9.7 Hz, 2H), 1.82 (q, J = 6.5, 5.9 Hz, 2H), 1.72 (d, J = 6.0 Hz, 2H), 1.30–1.22 (m, 10H), 1.19 (s, 8H), 0.88 (t, J = 6.7 Hz, 3H).

[0099] 5) Synthesis of Target Compound MNBS

[0100] Intermediate 1 (150 mg, 0.29 mmol) and Intermediate 4 (176 mg, 0.59 mmol) were dissolved in methanol and refluxed. Subsequently, silver carbonate (162 mg, 0.59 mmol) was added in portions. The reaction was terminated by TLC tracking until the raw materials disappeared. After cooling to room temperature, the solid was removed by suction filtration, and the solvent was removed by distillation under reduced pressure. The target compound (99 mg, yield: 49%) was obtained by column chromatography (dichloromethane:methanol, 100 - 10:1). 1 1H NMR (400 MHz, Methanol-d4) δ 8.39 (s, 1H), 8.04 (d, J = 9.6 Hz, 1H), 7.52 (dd, J = 9.7, 2.8 Hz, 1H), 7.42 (d, J = 2.8 Hz, 1H), 7.35 (s, 1H), 3.77 (q, J = 7.1 Hz, 4H), 3.68 (t, J = 6.8 Hz, 2H), 3.57 (td, J = 7.2, 3.7 Hz, 2H), 3.47–3.40 (m, 2H), 3.08 (s, 3H), 2.89 (td, J = 5.8, 2.4 Hz, 2H), 2.63 (t, J = 6.2 Hz, 2H), 2.21 (d, J = 6.0 Hz, 4H), 2.02–1.96 (m, 2H), 1.95–1.89 (m, 2H), 1.69 (s, 2H), 1.38 (s, 2H), 1.36 (s, 3H), 1.34 (s, 2H), 1.32–1.28 (m, 6H), 1.23 (s, 7H), 1.18 (s, 6H), 0.90 (t, J = 7.0 Hz, 5H). [M]+, calculated value: 699.4337, detected value: 699.4475.

[0101] Example 2

[0102] This example explores the spectral changes of the photosensitizer (MNBS) prepared in Example 1 in different solvents and the types of reactive oxygen species generated after light irradiation.

[0103] (1) Determination of the optical properties of the prepared photosensitizer

[0104] The solid MNBS was dissolved in DMSO to prepare a 10 mM stock solution for standby, and stored in the dark. The stock solution was diluted into different solvents (dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, ethyl acetate, water, acetone, toluene, methanol), and the final concentration of the photosensitizer in different solvent systems was 5 μM. The spectral data were recorded by ultraviolet and fluorescence spectrometers.

[0105] (2) Types of reactive oxygen species generated by the photosensitizer (MNBS) after light irradiation

[0106] Using 2′,7′-dichlorodihydrofluorescein (DCFH) as the reactive oxygen species scavenging reagent (excitation wavelength: 488 nm, emission wavelength: 500 - 640 nm); using singlet oxygen trapping reagent 1,3-diphenylisobenzofuran (DPBF) and methylene blue as references to determine the singlet oxygen generation ability of MNBS; using dihydroethidium (DHE) as the superoxide anion scavenging reagent (excitation wavelength: 561 nm, emission wavelength: 575 - 620 nm). The concentration of the photosensitizer was 5 μM, and the concentration of the reactive oxygen species scavenger was 5 μM. Under the irradiation of a 660 nm LED lamp at 3 mW / cm 2 the spectral changes at different time points were measured.

[0107] Experimental results: As Figure 4 (left) shows, the characteristic absorption peak of the photosensitizer MNBS is around 640 nm and the wavelength redshifts with the increase of polarity. In Figure 4 (right), it can be found that the characteristic emission peak of this photosensitizer in methanol is at 669 nm, located in the near-infrared region. As Figure 5 shown, compared with the light-only group, the fluorescence intensity of the photosensitizer plus light group gradually increases with the prolongation of the light irradiation time, indicating that the photosensitizer has a high reactive oxygen species generation ability. Further exploring the types of reactive oxygen species, as Figure 6 shown, the intensity of the maximum absorption peak of the DPBF light-only group has no obvious change, while the absorption of DPBF at 410 nm in the photosensitizer plus light group gradually weakens with the prolongation of the light irradiation time. Using MB as a reference, the singlet oxygen yield of MNBS was calculated to be 0.28. The detection results of superoxide anions are as Figure 7 shown, the fluorescence signal of the light-only group has no obvious change, and the fluorescence signal of DHE in the photosensitizer plus light group gradually increases. To sum up, the reactive oxygen species generated by MNBS include singlet oxygen and superoxide anions. The singlet oxygen yield of MNBS is as high as 0.28, which has exceeded most of the reported heavy-atom-free photosensitizers. At the same time, the superoxide anions generated by it can overcome the tumor hypoxic environment, making it still have a high photodynamic therapy efficiency under hypoxic conditions.

[0108] Example 3

[0109] This example studied the uptake ability of tumor cells to the photosensitizer prepared in Example 1, the localization ability on the cell membrane, and the cell killing ability evaluation.

[0110] (1) Uptake ability of tumor cells to the photosensitizer MNBS

[0111] In this example, mouse breast cancer cells were selected as the experimental target, and 1*10 5Cells were seeded in confocal dishes and cultured in an incubator until they reached the logarithmic growth phase. Then, the culture medium was removed and the cells were washed 2 - 3 times with PBS. The photosensitizer MNBS was diluted to 1 μM with culture medium and added to the dishes. The fluorescence signal was observed under a fluorescence confocal microscope (excitation wavelength: 640 nm, emission: 655 - 700 nm).

[0112] (2) Localization of photosensitizer in cells: 1×10 5 Cells were seeded in confocal dishes and cultured in an incubator until they reached the logarithmic growth phase. Then, the culture medium was removed and the cells were washed 2 - 3 times with PBS. The photosensitizer MNBS was diluted to 1 μM with culture medium and added to the dishes. Lysosome green fluorescence probe, mitochondrial green fluorescence probe, nuclear blue fluorescence probe, and cell membrane green fluorescence probe were added and co - incubated for 30 minutes. Then, the fluorescence signal was observed under a fluorescence confocal microscope (Lyso / Mito / DIO excitation wavelength: 488 nm, emission wavelength: 495 - 610 nm; Nucl excitation wavelength: 405 nm, emission wavelength: 420 - 500 nm; MNBS excitation wavelength: 640 nm, emission wavelength: 655 - 700 nm).

[0113] (3) Cytotoxicity and live - dead staining experiments: including phototoxicity and dark toxicity experiments. The specific operations are as follows:

[0114] Under normoxic conditions:

[0115] Phototoxicity experimental group: Three different types of tumor cells were seeded into 96 - well plates at a density of 10,000 cells per well and cultured in an incubator for 24 hours. Cells with poor adhesion and poor cell state were removed by washing with PBS. Different concentrations of the photosensitizer MNBS (0, 0.1, 0.2, 0.4, 0.6, 0.8, 1 μM) were added to each well, and 5 replicates were set for each concentration. Incubation was continued for half an hour and then irradiated (660 nm, 3 mW / cm 2 , 10 min). After 24 hours, the culture medium was removed, and 100 μL of 0.5 mg / mL MTT was added to each well. Four hours later, the liquid in the 96 - well plate was removed, and 100 μL of dimethyl sulfoxide was added. The absorbance at 490 nm was measured by an enzyme - linked immunosorbent assay (ELISA) reader to calculate the cell survival rate.

[0116] Dark toxicity experimental group: The experimental steps were as follows, but this group did not require irradiation. After adding the photosensitizer and incubating, the cells were cultured for another 24 hours, then MTT was added. Four hours later, dimethyl sulfoxide was added, and the absorbance at 490 nm was measured by an ELISA reader to calculate the cell survival rate.

[0117] Under hypoxic conditions:

[0118] Phototoxicity experimental group: The above 4 different types of tumor cells were placed on a 96-well plate and cultured in an incubator for 18 h, then cultured in an anaerobic bag (microaerophilic) for 6 h, and then different concentrations of MNBS were prepared. After exhausting the air, the cell culture medium containing the photosensitizer was added to the 96-well plate. The cells were again placed in an anaerobic bag with an oxygen content of about 2% and cultured for 0.5 h, and then irradiated under hypoxic conditions (660 nm, 5 mW / cm 2 ) for 10 min, and the light dose was detected by a densitometer through the 96-well plate and the sealing cover plate. After continuing to culture for 24 h, the culture medium was poured out, MTT was added, and after waiting for 4 h, the absorbance value of each well at 490 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the cell survival rate was calculated.

[0119] Dark toxicity experimental group: The experimental procedure was the same as that of the phototoxicity experimental group, but without light irradiation.

[0120] Live and dead cell staining: The experimental procedure was as follows; 1×10 5 cells were inoculated into a confocal dish and cultured in a cell incubator until the logarithmic growth phase of the cells. Then, the culture medium was poured out and the cells were washed 2-3 times with PBS and divided into four groups including a control group, a control plus light irradiation group, a photosensitizer group, and a photosensitizer plus light irradiation group. At the same time, the photosensitizer MNBS was diluted to 1 μM with the culture medium and added to the photosensitizer group and the photosensitizer plus light irradiation (light irradiation 0.5 h after addition), and after incubation for a period of time, Calcein-AM / PI was added to the four groups respectively, and the fluorescence signal was observed under a fluorescence confocal microscope. The excitation wavelength of Calcein-AM is 488 nm, and the receiving range is 500-540 nm. The excitation wavelength of PI is 535 nm, and the receiving range is 550-620 nm.

[0121] Experimental results: As Figure 8 shown in a, when the fluorescence signal intensity in the cells reached a plateau after 15 min of addition, and there was almost no difference in the intensity at 20 min compared with that at 15 min, indicating that MNBS can be rapidly taken up by tumor cells. As Figure 9 shown, the overlap of MNBS (red fluorescence) and the cell membrane fluorescence probe (green fluorescence) was very good, and only a small amount of red fluorescence was found in the cytoplasm, proving the significant cell membrane targeting ability of MNBS. As Figure 10 shown, mouse breast cancer cells (4T1), human breast cancer cells (MDA-MB-231, MCF-7), and human liver cancer cells (HepG2) were selected as experimental subjects. The dark toxicity of MNBS can be ignored, indicating its good in vitro biocompatibility. After light irradiation, the higher the concentration of the photosensitizer MNBS, the lower the cell survival rate. Under hypoxic conditions, a light dose of 3 J / cm 2 can also effectively kill tumor cells. To visualize the cell killing ability of MNBS, it was observed through a live and dead kit and a fluorescence confocal microscope. As Figure 8As shown in Figure b, bright green fluorescence signals were presented in the control group, the control plus light group, and the photosensitizer-only group, indicating that no damage was caused to tumor cells at this light dose or photosensitizer concentration. However, a bright red signal appeared in the photosensitizer plus light group, indicating that this photosensitizer has superior tumor cell killing ability.

[0122] From the above experimental results, it can be seen that under normoxic and hypoxic conditions, MNBS can generate reactive oxygen species to induce cell death at extremely low light doses.

[0123] Example 4

[0124] This example studied the induction of pyroptosis, immunogenic cell death, and evaluation of in vivo immune response by the photosensitizer MNBS.

[0125] Based on the previous cytotoxicity experiments, it was speculated that MNBS might activate inflammasomes by disrupting the permeability and instability of the plasma membrane, ultimately leading to immunogenic cell death. Therefore, further studies on the detection of markers related to MNBS-mediated immunogenic cell death were also carried out, as follows:

[0126] (1) Detection of pyroptosis and its markers

[0127] 10,000 4T1 cells were seeded in a confocal dish. After they grew to 80% confluence and completely adhered, they were washed 2 - 3 times with PBS, and then the photosensitizer MNBS (1 μM) diluted with DMEM was added. After incubation at room temperature for half an hour, light irradiation was performed, and the cell morphology was observed by confocal microscopy. Secondly, the content changes of the corresponding targets were detected by an ATP detection kit and a lactate dehydrogenase (LDH) detection kit.

[0128] (2) Detection of immunogenic cell death markers

[0129] Immunofluorescence staining was used to detect the immunogenic cell death marker high-mobility group protein 1 (HMGB1). 10,000 4T1 cells were seeded in a confocal dish and divided into four groups: Control group, Light group, MNBS group, and MNBS + Light group. After the cell density reached 80%, they were washed 2 - 3 times with PBS. The latter two groups were added with the photosensitizer MNBS (1 μM) diluted with DMEM. After incubation at room temperature for half an hour, the MNBS + Light group was irradiated with light (660 nm, 3 mW / cm 2, (10 min). Subsequently, the culture media of all four groups were poured out and washed 2 - 3 times with PBS. 1 mL of 4% paraformaldehyde was added to each confocal dish and fixed at 4°C for 20 min. After pouring out the fixative, PBS was added and shaken on a plate shaker for three washes, 5 min each time. After that, 1% Triton - 100 was added for permeabilization. After half an hour, it was poured out and washed three times with PBS. Blocking solution was added for half an hour and then washed three times with PBS. Then, the primary antibody (HMGB1, diluted 1000 - fold) was added and incubated overnight. The next day, the primary antibody dilution was removed and washed three times with PBS. Subsequently, the secondary antibody with fluorescent label (diluted 1000 - fold) was added and incubated at room temperature for two hours, then washed three times with PBS. 0.5 μL of DAPI dye (dissolved in DMSO, 5 mM) was added and the fluorescence signal was observed under a fluorescence confocal microscope.

[0130] (3) In vivo imaging and immunological analysis

[0131] Twenty female BALB / c mice (5 - 6 weeks old) were subcutaneously injected with 1×10 6 mouse breast cancer cells. When the tumor volume reached approximately 100 mm 3 , they were randomly divided into four groups, including a control group (injected with only 100 μL of normal saline), a control plus light group (injected with 100 μL of normal saline, irradiated with a 660 nm LED lamp at 50 mW / cm 2 , 10 min), a photosensitizer group (injected with only 100 μM of photosensitizer), and a photosensitizer plus light group (100 μM of photosensitizer and irradiated with a 660 nm LED lamp at 50 mW / cm 2 , 10 min). First, the photosensitizer was injected beside the tumor to observe the enrichment time of the photosensitizer in the in - vivo tumor to determine the optimal treatment time. Subsequently, according to the obtained optimal enrichment time of the tumor, photodynamic therapy was carried out. On the seventh day of treatment, some mice were sacrificed in each experimental group for anatomical analysis of the maturation of dendritic cells in the spleen and the activation of T cells in the tumor.

[0132] Experimental results: As Figure 11 shown in a, when the photosensitizer MNBS was added and irradiated, small vesicles (arrows in the figure) could be observed around the cell membrane at the 10th minute, and there were more and more with the extension of time, indicating that cell pyroptosis might be induced under light irradiation. Subsequently, the intracellular ATP content and extracellular LDH were measured. As Figure 11 shown in b, the intracellular ATP content gradually decreased with the increase in the concentration of the photosensitizer. As Figure 11 shown in c, the extracellular LDH concentration increased with the concentration of the photosensitizer. In summary, MNBS can cause cell pyroptosis under light irradiation. As Figure 12As shown, the marker of immunogenic cell death, HMGB1, was measured. There was no significant change in the HMGB1 signal in the nucleus of the control group, the light-only group, and the MNBS group, while the fluorescence signal in the cells of the photosensitizer MNBS plus light group was significantly weakened, indicating the migration of HMGB1. As Figure 13 shown, when the photosensitizer was injected beside the tumor, with the prolongation of time, the enrichment amount of the photosensitizer in the tumor continuously increased, reached the maximum at 4 h, and then gradually decayed, and the fluorescence almost disappeared after 24 h. This indicates that the photosensitizer molecule can rapidly enrich in tumor cells, and at the same time has a fast metabolism in the body and has good biosafety. Then, immune analysis was performed on mice with different treatments. As Figure 14 shown, the expression levels of CD86 and CD80 on mature dendritic cells in the MNBS+Light group were approximately 12 times that of the control group, and the content of CD8 + T cells in the tumor was approximately 2 times that of the control group.

[0133] The above results indicate that MNBS can cause pyroptosis under light, and then induce immunogenic cell death to improve the body's immune response and achieve photoimmunotherapy.

[0134] In summary, as described in the above examples, MNBS, as a new type of photosensitizer, not only has excellent photochemical properties and reactive oxygen species generation ability, but also shows cell membrane targeting, low cytotoxicity and immunomodulatory effects in tumor treatment, and has the potential to be developed into an efficient photodynamic therapy drug. Its properties include:

[0135] 1. Optical properties and reactive oxygen species generation ability:

[0136] MNBS exhibits stable optical properties in different solvents, with a characteristic absorption peak at about 640 nm, and a red shift occurs with the increase of solvent polarity, indicating its good solubility and photostability.

[0137] Under light illumination, MNBS can efficiently generate singlet oxygen with a yield as high as 0.28, which is superior to most known heavy-atom-free photosensitizers, showing its excellent photochemical performance.

[0138] MNBS can also generate superoxide anions, and this property enables it to maintain an efficient photodynamic therapy effect in the hypoxic environment of tumors.

[0139] 2. Tumor cell uptake and cell membrane targeting ability:

[0140] Tumor cells can rapidly uptake MNBS, which is beneficial for its accumulation at the tumor site, and MNBS specifically localizes to the tumor cell membrane and can effectively damage the cell membrane under light illumination, improving the treatment specificity.

[0141] 3. Cytotoxicity and phototoxicity:

[0142] MNBS has low cytotoxicity to cells under dark conditions, indicating its good biocompatibility in vitro.

[0143] Under light illumination, MNBS shows obvious cytotoxicity. The higher the concentration, the lower the cell survival rate, indicating its significant photodynamic effect.

[0144] 4. Pyroptosis and immunogenic cell death:

[0145] MNBS can induce pyroptosis, manifested as the formation of vesicles on the cell membrane, a decrease in intracellular ATP levels, and an increase in LDH release.

[0146] MNBS can also promote immunogenic cell death, such as the translocation of HMGB1, which may activate the body's immune system and produce a long-term inhibitory effect on tumors.

[0147] 5. In vivo application and immune response:

[0148] After peritumoral injection, MNBS rapidly accumulates in tumor tissues, indicating its good tumor targeting ability.

[0149] After treatment, MNBS can improve the maturation of dendritic cells in the tumor site and the expression of CD8 + T cells in the spleen, activate the body's immune response, and achieve the effect of photoimmunotherapy.

[0150] It should be understood that the above embodiments are only for more clearly illustrating the technical solutions of the present invention, rather than limiting the scope of its protection. Those of ordinary skill in the art can make various modifications and variations to the above embodiments without departing from the spirit and basic principles of the present invention, but these modifications and variations still fall within the protection scope of the claims of the present invention and their equivalent replacements. The protection scope of the present invention should be subject to the appended claims, rather than being limited to the specific details of the above embodiments.

Claims

1. A phenothiazine cell membrane-targeted photosensitizer, the photosensitizer having the structure shown in the following formula I: The R is selected from one of dimethylamino, diethylamino, aziridine, azetidine, azolidine, and cyclohexylamine.

2. The photosensitizer according to claim 1, characterized in that: The photosensitizer of formula 1 is selected from one of C1, C2, C3, C4, C5, and C6; 3. The preparation method of the photosensitizer according to claim 1, characterized in that: It includes the following steps: 1) In an aqueous solution of aluminum sulfate, an aniline derivative is mixed with sodium thiosulfate and zinc chloride, an aqueous solution of potassium dichromate is added, stirred at low temperature, and after the reaction is completed, filtered and the precipitate is washed with organic solvent I. The crude product is refluxed in methanol and filtered to obtain intermediate 1; The aniline derivative is N,N-dimethyl-p-phenylenediamine, 4-aziridinylaniline, 4-azetidinylaniline, 4-azolidinylaniline, 4-cyclohexylaniline; 2) 1-Amino-5,6,7,8-tetrahydronaphthylamine, 1-bromo-3-chloropropane, and potassium carbonate are refluxed in organic solvent II, and intermediate 2 is purified and separated; 3) Intermediate 2, N-methyl-1-dodecylamine, potassium iodide, and potassium carbonate are refluxed in organic solvent III, and intermediate 3 is purified and separated; 4) Intermediate 3 and 1,3-propane sultone are refluxed in organic solvent IV, and intermediate 4 is purified and separated; 5) Intermediate 4, intermediate 1, and silver carbonate are refluxed in organic solvent V, and the target product is purified and separated.

4. The method according to claim 3, wherein: In step 1), the molar ratio of the aniline derivative, aluminum sulfate, sodium thiosulfate, zinc chloride, and potassium dichromate is 1:1-2:2-3:1-2:0.1-0.

5.

5. The method according to claim 3, wherein: In step 1), the concentration range of the aqueous solution of potassium dichromate is 0.1-1 mM.

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

7. The method according to claim 3, wherein: In step 3), the molar ratio of intermediate 2, N-methyl-1-dodecylamine, potassium iodide, and potassium carbonate is 1:1-2:1-2:1-2.

8. The method according to claim 3, wherein: In step 4), the molar ratio of intermediate 3 and 1,3-propane sultone is 1:1-2.

9. The method according to claim 3, wherein: In step 5), the molar ratio of intermediate 4, intermediate 1, and silver carbonate is 1:1-3:1-2.

10. Use of the photosensitizer according to claim 1 in the preparation of preparations for non-disease diagnosis and treatment directions, including the preparation of drugs for photodynamic therapy, tumor immunotherapy, and preparations for pyroptosis research.

Images