Preparation method and application of endoplasmic reticulum targeted pyroptosis-promoting near-infrared dye

Abstract

The invention discloses a near-infrared photosensitizer for promoting pyroptosis in an endoplasmic reticulum targeting manner. The near-infrared photosensitizer comprises a preparation process of the near-infrared photosensitizer and a tumor photoimmunotherapy effect combined with photothermal and photodynamic effects. According to the invention, heptamethine cyanine molecules are modified by the photosensitizer, and the introduced p-toluene sulfonamide group and tetraphenyl ethylene (TPE) group can specifically recognize a sulfanilamide receptor on an endoplasmic reticulum membrane and specifically target to the endoplasmic reticulum of a cell, so that the photo-thermal stability and ROS (reactive oxygen species) generation efficiency of the photosensitizer are enhanced. According to the photosensitizer disclosed by the invention, different groups or probes can be introduced to the other end of the TPE, and when R is equal to H, the photosensitizer is IR780T-ER disclosed by the invention; and when R is equal to IR780-ER, the photosensitizer is T780T-ER disclosed by the invention. The photosensitizer has cell endoplasmic reticulum targeting and accumulation effects, and endoplasmic reticulum stress can be caused under near-infrared light irradiation. The photosensitizer disclosed by the invention has the effect of enhancing the tumor immunotherapy effect by inducing endoplasmic reticulum stress of tumor cells to generate immunogenic death and triggering pyroptosis of the cells. The photosensitizer disclosed by the invention is expected to be applied to photoimmunotherapy of clinical tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent biological diagnosis and treatment, and specifically relates to the preparation of a near-infrared photosensitizer for endoplasmic reticulum-targeted cell pyroptosis and a photoimmunotherapy combining photothermal and photodynamic therapy. Background Art

[0002] Cancer immunotherapy stimulates or enhances the body's own immune system to enable it to recognize and attack cancer cells. Unlike traditional radiotherapy and chemotherapy, immunotherapy focuses more on restoring and utilizing the patient's own immune capacity to achieve the purpose of treatment. Tumor antigen-based immunotherapy has also shown satisfactory response rates in various cancers, including vaccines based on tumor-specific antigens, personalized vaccines, whole cancer cell vaccines, and new antigen-targeted vaccines. DC vaccines also show the ability to induce antigen-specific immune responses in vivo. However, in the face of patient heterogeneity, there are still huge limitations, such as uncertain doses and characteristics, as well as expensive costs. Phototherapy, using photosensitizers to convert light energy into reactive oxygen species (ROS) or heat energy under near-infrared (NIR) light irradiation, while high temperature and ROS can induce immunogenic cell death (ICD) of tumor cells, which produce proinflammatory cytokines and damage-associated molecular patterns (DAMPs), leading to anti-tumor immune responses. As a more specific and simpler method, it is expected to strengthen the current cancer immunotherapy strategy and improve its efficacy and safety.

[0003] Pyroptosis is a lytic and inflammatory form of cell death different from apoptosis, and is believed to enhance the ICD process of tumor cells. Therefore, the development of new photosensitizers to trigger pyroptosis to improve the efficiency of immunogenic cell death at the tumor site is considered an important research direction for cancer immunotherapy. The endoplasmic reticulum is the largest organelle in eukaryotic cells and plays an important role in protein synthesis, folding, post-translational modification, and calcium homeostasis. Studies have shown that endoplasmic reticulum stress can stimulate a greater degree of cell pyroptosis, and endoplasmic reticulum stress caused by ROS has been shown to be one of the causes of immunogenic cell death. Designing photosensitizers that specifically target the endoplasmic reticulum of cells and destroying the endoplasmic reticulum function through PDT / PTT can better trigger ICD and enhance anti-tumor immune responses. Heptamethine cyanine dyes have excellent light absorption in the NIR region (>700 nm), deep tissue penetration, easy synthesis and low biological toxicity. Introducing the p-toluenesulfonamide group into the heptamethine cyanine molecule can enhance its endoplasmic reticulum targeting ability, while improving its stability and phototherapeutic effect, which is of great research significance for tumor immunotherapy. Summary of the invention

[0004] The purpose of the present invention is to provide a cyanine photosensitizer molecule with endoplasmic reticulum targeting, which can trigger cell pyroptosis and enhance immunogenic cell death. Among them, the photosensitizer T780T-ER has good endoplasmic reticulum targeting ability and high type I and type II ROS generation ability; in vitro, it can cause tumor cell immunogenic death and enhance the release of inflammatory factors and damage-related molecular patterns through cell pyroptosis. Effective anti-tumor immune response is shown in mice.

[0005] The present invention provides a heptamethine cyanine photosensitizer molecule into which a cell endoplasmic reticulum targeting group is introduced, and its structural formula (1) is as follows: (1) Where R is H, CH 3 , C 2 H 5 , CN or any photosensitizer molecule. When R=H, it is the photosensitizer IR780T-ER of the present invention; When R=IR780-ER, it is the photosensitizer T780T-ER of the present invention.

[0006] The synthesis steps of the photosensitizer provided by the present invention are shown in the attached Figure 1 .

[0007] The present invention provides a method for preparing a photosensitizer, comprising the following steps: Under nitrogen protection, with anhydrous acetonitrile as solvent, 3-bromopropylamine and 2,3,3-trimethylindole were stirred and refluxed at 120 °C for 10 h. The solvent was evaporated to dryness, and 1,4-dioxane and methanol were recrystallized to obtain compound 1. Then, under nitrogen protection, with n-butanol / toluene (7 / 3, V / V) as solvent, compound 1 and 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde were stirred and refluxed at 120 °C for 16 h. The solvent was evaporated to dryness, and n-hexane and methanol were recrystallized to obtain compound 2.

[0008] IR780-ER: Under nitrogen protection, anhydrous acetonitrile was used as solvent, compound 2 and triethylamine were stirred at room temperature for 4 h. The solvent was evaporated and the crude product was purified by silica gel chromatography to prepare compound IR780-ER; IR780T-ER: Under nitrogen protection, anhydrous DMF was used as solvent, IR780-ER / 1,2-diphenyl-1,2-di(4-hydroxyphenyl)ethylene (1:4) and potassium carbonate were stirred at room temperature for 48 h. The solvent was evaporated and the crude product was purified by silica gel chromatography to prepare compound IR780T-ER; T780T-ER: Under nitrogen protection, anhydrous DMF was used as solvent, IR780-ER / 1,2-diphenyl-1,2-di(4-hydroxyphenyl)ethylene (3:1) and potassium carbonate were stirred at room temperature for 48 h. The solvent was evaporated and the crude product was purified by silica gel chromatography to prepare compound T780T-ER.

[0009] Compared with the prior art, the invention has the following beneficial effects.

[0010] The photosensitizer of the present invention introducing p-toluenesulfonamide has the following characteristics: (1) The photosensitizer of the present invention modifies the alkyl chain connected to the nitrogen atom on the indole ring of the heptamethine cyanine molecule and introduces two molecules of p-toluenesulfonamide groups, which can specifically recognize the sulfonamide receptor on the endoplasmic reticulum membrane and specifically target the endoplasmic reticulum of the cell; (2) The photosensitizer T780T-ER of the present invention introduces a tetraphenylethylene (TPE) group to enhance its photostability and ROS generation efficiency; (3) The photosensitizer T780T-ER of the present invention can target the endoplasmic reticulum of the cell and generate type I and type II ROS in situ to cause endoplasmic reticulum stress and promote the immunogenic death of tumor cells; (4) The photosensitizer T780T-ER of the present invention can trigger cell pyroptosis after 808 nm laser irradiation, releasing inflammatory factors and DAMPs; (5) The photosensitizer T780T-ER of the present invention effectively kills primary tumors in tumor-bearing mice, triggers immune responses, and inhibits the growth of distant tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The invention discloses a synthetic route for the photosensitizer.

[0012] Figure 2 The UV absorption graph and fluorescence emission graph of the photosensitizer (10 µM) of the present invention in DMSO and aqueous solution, respectively.

[0013] Figure 3 It is a comparison diagram of type I and type II ROS generation of the photosensitizer of the present invention, a photothermal curve diagram and a photothermal cycle diagram.

[0014] Figure 4 The confocal microscopic images show that the photosensitizer of the present invention co-localizes with ER-Tracker Blue and Mito-Tracker Green in A549 cells.

[0015] Figure 5 The figures are cell morphology pictures of cell pyroptosis induced by the photosensitizer T780T-ER of the present invention in A549 cells and 4T1 cells after illumination, and protein immunoblotting pictures of 4T1 cells.

[0016] Figure 6The immunofluorescence images of CRT and HMGB1 proteins in 4T1 cells before and after illumination of the photosensitizer T780T-ER of the present invention.

[0017] Figure 7 These are the photothermal images and therapeutic effect diagrams of the photosensitizer T780T-ER of the present invention in phototherapy in 4T1 tumor-bearing mice. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described with the following specific embodiments, but the present invention is by no means limited to these examples. The following description is only a preferred embodiment of the present invention, which is only used to explain the present invention and cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that any modification, substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0019] The photosensitizer of the present invention and the specific implementation of its preparation method and application are further described below in conjunction with the accompanying drawings and examples.

[0020] Example 1.

[0021] Synthesis of probe IR780-ER.

[0022]

[0023] The synthesis method is as follows.

[0024] Specific steps: Place the magneton, compound 1 (0.4 g, 2.32 mol) and 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde (1.12 g, 5.15 mol) in a 100 mL two-necked flask, assemble a reflux device, replace the vacuum-nitrogen atmosphere three times, add 50 mL of n-butanol / toluene (7 / 3, V / V) with a syringe, heat to 120°C and reflux for 16 hours. Evaporate the solvent, and recrystallize from n-hexane and methanol to obtain compound 2. Place the magneton, compound 2 (30 mg, 0.05 mmol), and p-toluenesulfonyl chloride (17.6 mg, 0.09 mmol) in a 10 mL two-necked flask, assemble a reflux device, replace the vacuum-nitrogen atmosphere three times, and add 3 mL of anhydrous acetonitrile and 0.2 mL of triethylamine with a syringe. Stir at room temperature for 4 h under nitrogen protection. TLC detection (developing agent CH 2 Cl 2 :CH 3 OH=15:1) After the reaction is complete, the solvent is evaporated with an oil pump, and the crude product is separated by silica gel chromatography. 2 Cl 2 :CH 3The product was eluted with OH=30:1 eluent to obtain green solid compound IR780-ER. 1 H NMR (400 MHz, Methanol- d 4 ) δ 8.33 (d, J =14.1 Hz, 2H), 7.60 (d, J = 8.2 Hz, 4H), 7.42 (d, J = 7.4 Hz, 2H), 7.32 (t, J = 7.6Hz, 3H), 7.27 – 7.17 (m, 9H), 6.25 (d, J = 14.1 Hz, 2H), 4.20 – 4.11 (m, 4H), 2.85 (t, J = 6.4 Hz, 4H), 2.67 (s, 4H), 2.56 (s, 4H), 2.30 (s, 4H), 1.88 (d, J =8.0 Hz, 4H), 1.62 (s, 12H). HRMS (ESI) m / z: [M] + , calculated, 877.3583; actual value, 877.3582.

[0025] Synthesis of probe IR780T-ER.

[0026] The synthesis method is as follows.

[0027]

[0028] Specific steps: Place the magneton, IR780-ER (50 mg, 0.052 mmol), 1,2-di(4-hydroxyphenyl)-1,2-diphenylethylene (75 mg, 0.208 mmol) and potassium carbonate (22 mg, 0.156 mmol) in a 10 mL two-necked flask, assemble a reflux device, replace the vacuum-nitrogen atmosphere three times, and then add 2 mL of DMF with a syringe. Stir at room temperature for 48 h under nitrogen protection. TLC detection (developing solvent CH 2 Cl 2 :CH 3 OH=15:1) After the reaction is complete, the solvent is evaporated with an oil pump, and the crude product is separated by silica gel chromatography. 2 Cl 2 :CH 3 The product was eluted with OH=40:1 eluent to obtain a green solid compound IR780T-ER. 1H NMR (400MHz, Chloroform- d ) δ 7.76 (d, J = 8.1 Hz, 8H), 7.23 (s, 2H), 7.16 (d, J = 8.2 Hz, 4H), 7.12 (s, 2H), 7.06 – 6.97 (m, 10H), 6.93 (s, 2H), 6.87 (d, J = 7.2 Hz,2H), 6.77 (d, J = 8.4 Hz, 2H), 6.70 (d, J = 8.2 Hz, 2H), 6.54 (s, 2H), 6.05 (d, J =10.6 Hz, 2H), 4.12 (s, 4H), 3.01 (s, 4H), 2.60 (s, 4H), 2.32 (s, 6H), 1.90(d, J = 47.1 Hz, 6H), 1.34 (s, 12H). HRMS (ESI) m / z: [M] + , calculated, 1205.5279; actual value, 1205.5283.

[0029] Synthesis of probe T780T-ER.

[0030]

[0031] The synthesis method is as follows.

[0032] Specific steps: Place the magnetron, IR780-ER (20 mg, 0.02 mmol), 1,2-di(4-hydroxyphenyl)-1,2-diphenylethylene (2.53 mg, 0.007 mmol) and potassium carbonate (6 mg, 0.02 mmol) in a 10 mL two-necked flask, assemble a reflux device, replace the vacuum-nitrogen atmosphere three times, and then add 2 mL of DMF with a syringe. Stir at room temperature for 48 h under nitrogen protection. TLC detection (developing solvent CH 2 Cl 2 :CH 3 OH=15:1) After the reaction is complete, the solvent is evaporated with an oil pump, and the crude product is separated by silica gel chromatography. 2 Cl 2 :CH 3 The product was eluted with OH=20:1 eluent to obtain green solid compound T780T-ER. 1H NMR (400 MHz, Methanol- d 4 ) δ 7.66 (d, J = 8.1 Hz, 12H), 7.35 (d, J = 7.6 Hz, 6H), 7.27 (t, J =9.0 Hz, 22H), 6.97 (d, J = 8.2 Hz, 4H), 6.90 (d, J = 8.0 Hz, 8H), 6.72 (d, J = 6.5Hz, 6H), 6.02 – 5.91 (m, 4H), 4.22 (s, 8H), 2.89 (s, 8H), 2.32 (s, 10H), 1.85(s, 8H), 1.26 (s, 6H), 1.17 (d, J = 11.4 Hz, 32H). HRMS (ESI) m / z: [M / 2] + , calculated, 1023.9564; actual value, 1023.9578.

[0033] Example 2.

[0034] UV-visible absorption spectrum and fluorescence absorption spectrum: 10 µM IR780-ER, IR780T-ER and T780T-ER were prepared in DMSO and aqueous solution respectively, and the fluorescence spectrum and UV absorption spectrum were measured. The results are as follows: Figure 2 .

[0035] Example 3.

[0036] ROS generation and photothermal stability: Prepare 2 mL of 5 µM aqueous solution of IR780-ER, IR780T-ER and T780T-ER, add 10 µM DHR123 solution, 10 µM HPF solution or 50 µM ABDA solution, and mix well. 2) for 10 minutes, and the fluorescence spectrum or UV absorption spectrum of the solution was recorded at corresponding time intervals, and the fluorescence emission intensity-time at 525 nm (DHR123) and 515 nm (HPF) and the UV absorption intensity-time at 378 nm (ABDA) were plotted. 2 mL of DMSO solution of 10 µM IR780-ER, IR780T-ER and T780T-ER were prepared, respectively, and irradiated with 808 nm laser of different powers (0.3, 0.5, 0.8 W / cm 2 ), record the temperature value every 30 seconds, and record the temperature change from 0 to 10 min. Prepare 2 mL of DMSO solution of 10 μM IR780-ER, IR780T-ER and T780T-ER respectively, and irradiate with 808 nm laser (power 0.3 W / cm 2 ), record the temperature value every 30 seconds, record the temperature change from 0 to 10 minutes, remove the laser after 10 minutes, let the solution cool naturally, record the temperature value every 30 seconds, and record the color of the solution after the solution temperature cools naturally to the initial temperature. Repeat the above operation four times and draw a temperature-time curve. The results are as follows Figure 3 .

[0037] Example 4.

[0038] Fluorescence confocal laser scanning microscopy imaging: A549 cells were seeded in a 35 mm glass bottom culture dish (special for confocal microscope) at a density of 3*10 5 / dish, and culture overnight. Probes IR780-ER (5 µM), IR780T-ER (10 µM) and T780T-ER (10 µM) were added for 5 hours, and then 100 nM ER-tracker Blue or Mito-tracker Green was added and cultured for another half an hour. The culture medium was aspirated and washed with PBS three times. The cell fluorescence was recorded by confocal microscopy. The results are shown in Figure 4 .

[0039] Example 5.

[0040] Cell morphology and western blot evaluation of cell pyroptosis: A549 and 4T1 cells were seeded in 35 mm glass-bottom culture dishes (for confocal microscopy) at a density of 3*10 5 / dish, and cultured overnight. The original culture medium was discarded, and the culture medium containing 5 μM probe T780T-ER was added and cultured for 24 h. The laser irradiation group was irradiated with 808 nm laser (power 0.3 W / cm 2) for 5 minutes, and then cultured for another 6 hours before observing cell fluorescence images using a confocal microscope. Protein immunoblotting: 4T1 cells were seeded in 96-well plates at a density of 200,000 cells / well and cultured for 24 hours. The original culture medium was discarded, and different groups were set up. Culture medium containing 5 µM probe T780T-ER was added and cultured for 24 hours. The laser irradiation group was irradiated with 808 nm laser (power 0.3 W / cm2) for 5 minutes. After culturing for another 6 hours, the cells were collected and total protein was extracted with lysis buffer. The extracted proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. Nonspecific sites were blocked with skim milk (10%). After incubation with diluted primary antibodies (caspase-3, GSDME, β-actin antibody) at 4°C overnight, the PVDF membrane was treated with HRP-conjugated secondary antibodies, and finally a chemiluminescence system was used to provide immunoassay readings. The results are shown in the figure. Figure 5 .

[0041] Example 6.

[0042] Immunofluorescence confocal laser scanning microscopy imaging of CRT and HMGB1 proteins: 4T1 cells were seeded in a 35 mm glass bottom culture dish (special for confocal microscope) at a density of 3*10 5 / dish, and cultured overnight. The original culture medium was discarded, and different groups were set up. The culture medium containing 5 μM probe T780T-ER was added and cultured for 24 h. The laser irradiation group was irradiated with 808 nm laser (power 0.3 W / cm 2 ) for 5 minutes, and then after 6 hours of culture, the cells were fixed with 4% paraformaldehyde for 20 minutes, blocked with 5% BSA for 0.5 hours, and then incubated with diluted CRT or HMGB1 (permeabilized with 0.1% Triton-100 for 5 minutes) antibodies at 4°C overnight. Finally, the cells were incubated with Alexa Fluor 488-labeled secondary antibodies for 2 hours and stained with DAPI for 10 minutes. After rinsing with PBS three times, the cell fluorescence images were observed with a confocal microscope. The results are shown in Figure 6 .

[0043] Example 7.

[0044] In vivo photothermal evaluation and tumor treatment: 1 × 10 7 4T1 cancer cells were implanted subcutaneously into the right side of each BALB / c mouse as a primary tumor. Five days later, the same number of 4T1 cells were implanted into the left abdomen of the mouse as a distal tumor. When the primary tumor of the mouse reached 100 mm 3At 9 and 24 hours later, the mice were divided into five groups (n = 6). Three groups of mice were injected intratumorally with T780T-ER (100 µM, 100 µL), and the other two groups were injected with PBS (100 µL) as a control group. 2 or 0.8 W / cm 2 The primary tumors of two mice in the T780T-ER group and the PBS group were irradiated with 10% irradiation for 10 minutes and the tumor temperature was recorded with a thermal imager, and then the treatment was repeated twice. The tumor volume was measured every other day and calculated as follows: Volume = (tumor length) × (tumor width) 2 / 2. After 21 days, all mice were killed by cervical dislocation, and the main organs were collected for H&E staining. Tumor tissues and spleens of five groups of mice (n=3) were used for FACS analysis, and residual tumor tissues were fixed with 4% formaldehyde solution for IHC and IF analysis.

Claims

1. A near-infrared photosensitizer that targets the endoplasmic reticulum and promotes cell pyroptosis, the general structural formula (1) of which is as follows: (1)。 2. The cyanine photosensitizer according to claim 1, characterized in that: The photosensitizer of the present invention modifies the heptamethine cyanine molecule, and the introduced p-toluenesulfonamide group and tetraphenylethylene (TPE) group can specifically recognize the sulfonamide receptor on the endoplasmic reticulum membrane, specifically target the endoplasmic reticulum of the cell, and enhance the photothermal stability and ROS generation efficiency of the photosensitizer.

3. The photosensitizer according to claim 1, wherein R is a group such as H, CH3, C2H5, CN, or any photosensitizer molecule. When R=H, it is the photosensitizer IR780T-ER of the present invention; when R=IR780-ER, it is the photosensitizer T780T-ER of the present invention. .

4. The photosensitizer according to claim 1 has the effect of targeting and accumulating in the endoplasmic reticulum of cells, and can cause endoplasmic reticulum stress under near-infrared light irradiation.

5. The photosensitizer as claimed in claim 1 produces immunogenic death through endoplasmic reticulum stress of cells and triggers cell pyroptosis to enhance the effect of tumor immunotherapy.

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