Method for preparing endoplasmic reticulum-targeted pro-apoptotic near-infrared dye and applications thereof

Abstract

The application discloses a near-infrared photosensitizer for promoting pyroptosis of endoplasmic reticulum, a preparation process thereof and a tumor photoimmunotherapy effect combining photothermal and photodynamic power. The photosensitizer is modified on a heptamethine cyanine molecule, and a p-toluenesulfonamide group and a tetraphenylethylene (TPE) group are introduced, so that the photosensitizer can specifically recognize sulfamide receptors on an endoplasmic reticulum membrane, is specifically targeted to the endoplasmic reticulum of cells, and the photothermal stability and ROS generation efficiency of the photosensitizer are enhanced. The photosensitizer can introduce different groups or probes to the other end of the TPE, and when R=H, the photosensitizer is IR780T-ER; when R=IR780-ER, the photosensitizer is T780T-ER. The photosensitizer has the effects of endoplasmic reticulum targeting and accumulation in cells, and can cause endoplasmic reticulum stress under near-infrared light irradiation. The photosensitizer can produce immunogenic death by causing endoplasmic reticulum stress of tumor cells, and trigger pyroptosis, so as to enhance the effect of tumor immunotherapy. The photosensitizer disclosed by the application is expected to be applied to photoimmunotherapy of clinical tumors.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence bio-diagnostic and therapeutic technology, specifically the preparation of a near-infrared photosensitizer that targets endoplasmic reticulum to promote pyroptosis and photoimmunotherapy combining photothermal and photodynamic therapies. Background Technology

[0002] Cancer immunotherapy works by stimulating or enhancing the body's own immune system, enabling it to recognize and attack cancer cells. Unlike traditional radiotherapy and chemotherapy, immunotherapy focuses on restoring and utilizing the patient's own immune capacity to achieve therapeutic goals. Tumor antigen-based immunotherapies have shown satisfactory response rates in various cancers, including vaccines based on tumor-specific antigens, personalized vaccines, whole-cell cancer cell vaccines, and neoantigen-targeted vaccines. DC vaccines have also shown the ability to induce antigen-specific immune responses in vivo. However, significant limitations remain due to patient heterogeneity, such as uncertain dosage and characteristics, and high costs. Phototherapy utilizes photosensitizers to convert light energy into reactive oxygen species (ROS) or heat energy under near-infrared (NIR) light irradiation. High temperatures and ROS can induce immunogenic cell death (ICD) in tumor cells, producing pro-inflammatory cytokines and damage-associated molecular patterns (DAMPs), leading to an anti-tumor immune response. As a more specific and simpler approach, it holds promise for enhancing current cancer immunotherapy strategies and improving their efficacy and safety.

[0003] Pyroptosis, a form of lytic and inflammatory cell death distinct from apoptosis, is believed to enhance the immunogenic cell death (ICD) process in tumor cells. Therefore, developing novel photosensitizers to trigger pyroptosis and improve the efficiency of immunogenic cell death at tumor sites is considered an important research direction in cancer immunotherapy. The endoplasmic reticulum (ER), the largest organelle in eukaryotic cells, plays a crucial role in protein synthesis, folding, post-translational modifications, and calcium homeostasis. Studies have shown that ER stress can induce greater pyroptosis, and ROS-induced ER stress has been proven to be one of the causes of immunogenic cell death. Designing photosensitizers that specifically target the ER and disrupting ER function through PDT / PTT can better trigger ICD and enhance anti-tumor immune responses. Heptamethrin dyes exhibit excellent light absorption, deep tissue penetration, ease of synthesis, and low biotoxicity in the NIR region (>700 nm). Introducing p-toluenesulfonamide groups into heptamethrin molecules can enhance their endoplasmic reticulum targeting ability, while also improving their stability and phototherapy efficacy, which is of great research significance for tumor immunotherapy. Summary of the Invention

[0004] The purpose of this invention is to provide a cyanin photosensitizer molecule with endoplasmic reticulum targeting activity that can trigger pyroptosis and enhance immunogenic cell death. The photosensitizer T780T-ER exhibits good endoplasmic reticulum targeting ability and high generation capacity of type I and type II ROS; in vitro, it can induce immunogenic cell death in tumor cells and enhance the release of inflammatory factors and damage-related molecular patterns through pyroptosis. In vivo, it demonstrates an effective anti-tumor immune response in mice.

[0005] This invention provides a heptamethrin photosensitizer molecule with an endoplasmic reticulum targeting group introduced into the cell, the structural formula of which is shown below (1): (1)

[0006] 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 this invention; when R=IR780-ER, it is the photosensitizer T780T-ER of this invention.

[0007] The photosensitizer synthesis steps provided by this invention are shown in the appendix. Figure 1 .

[0008] This invention provides a method for preparing a photosensitizer, comprising the following steps:

[0009] Under nitrogen protection, 3-bromopropylamine and 2,3,3-trimethylindole were stirred and refluxed at 120 °C for 10 h using anhydrous acetonitrile as solvent. The solvent was evaporated, and 1,4-dioxane and methanol were recrystallized to give compound 1. Then, under nitrogen protection, compound 1 and 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde were stirred and refluxed at 120 °C for 16 h using n-butanol / toluene (7 / 3, V / V) as solvent. The solvent was evaporated, and n-hexane and methanol were recrystallized to give compound 2.

[0010] IR780-ER: Compound 2 and triethylamine were mixed under nitrogen protection and stirred at room temperature for 4 h using anhydrous acetonitrile as solvent. The solvent was evaporated, and the crude product was purified by silica gel chromatography to prepare compound IR780-ER.

[0011] IR780T-ER: Under nitrogen protection, using anhydrous DMF 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.

[0012] T780T-ER: Under nitrogen protection, using anhydrous DMF 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 obtain compound T780T-ER.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0014] The photosensitizer of p-toluenesulfonamide introduced in this invention has the following characteristics: (1) The photosensitizer of this invention modifies the alkyl chain attached to the nitrogen atom on the indole ring of the p-toluenesulfonamide molecule, introducing two molecules of p-toluenesulfonamide group, which can specifically recognize sulfonamide receptors on the endoplasmic reticulum membrane and specifically target the endoplasmic reticulum; (2) The photosensitizer T780T-ER of this invention introduces a tetraphenylethylene (TPE) group to enhance its photostability and ROS generation efficiency; (3) The photosensitizer T780T-ER of this invention can target the endoplasmic reticulum and generate type I and type II ROS in situ, causing endoplasmic reticulum stress and promoting immunogenic death of tumor cells; (4) The photosensitizer T780T-ER of this invention can trigger pyroptosis after irradiation with 808 nm laser, releasing inflammatory factors and DAMPs; (5) The photosensitizer T780T-ER of this invention effectively kills the primary tumor in tumor-bearing mice and triggers an immune response, inhibiting the growth of distant tumors. Attached Figure Description

[0015] Figure 1 This is the synthetic route for the photosensitizer of the present invention.

[0016] Figure 2 The images show the UV absorption and fluorescence emission of the photosensitizer (10 µM) of this invention in DMSO and aqueous solution, respectively.

[0017] Figure 3 The diagram shows a comparison of type I and type II ROS generation, along with photothermal curves and photothermal cycling diagrams for the photosensitizers of this invention.

[0018] Figure 4 This is a confocal microscopy image showing the co-localization of the photosensitizer of this invention with ER-Tracker Blue and Mito-Tracker Green in A549 cells.

[0019] Figure 5 The images show cell morphology diagrams of A549 and 4T1 cells induced by light irradiation of the photosensitizer T780T-ER of this invention, as well as Western blot images of 4T1 cells.

[0020] Figure 6This is an immunofluorescence image of CRT and HMGB1 proteins in 4T1 cells before and after light exposure using the photosensitizer T780T-ER of this invention.

[0021] Figure 7 Photothermal images and therapeutic effects of the photosensitizer T780T-ER of this invention in 4T1 tumor-bearing mice. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is illustrated by the following specific embodiments, but the invention is by no means limited to these examples. The following descriptions are merely preferred embodiments of the invention and are used only to explain the invention; they should not be construed as limiting the scope of the invention. It should be noted that any modifications, substitutions, or improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

[0023] The following description, in conjunction with the accompanying drawings and examples, further illustrates the specific implementation of the photosensitizer of the present invention, its preparation method, and its application.

[0024] Example 1.

[0025] Synthesis of probe IR780-ER.

[0026]

[0027] The synthesis method is as follows.

[0028] Specific steps: A magnetic flask, compound 1 (0.4 g, 2.32 mol), and 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde (1.12 g, 5.15 mol) were placed in a 100 mL two-necked flask. A reflux apparatus was assembled, and the mixture was purged three times under vacuum and nitrogen. 50 mL of n-butanol / toluene (7 / 3, V / V) was added using a syringe, and the mixture was heated to 120°C and refluxed for 16 hours. The solvent was evaporated, and the mixture was recrystallized from n-hexane and methanol to obtain compound 2. A magnetic flask, compound 2 (30 mg, 0.05 mmol), and p-toluenesulfonyl chloride (17.6 mg, 0.09 mmol) were placed in a 10 mL two-necked flask. A reflux apparatus was assembled, and the mixture was purged three times under vacuum and nitrogen. 3 mL of anhydrous acetonitrile and 0.2 mL of triethylamine were added using a syringe. The mixture was stirred at room temperature for 4 hours under nitrogen protection. After the reaction was complete by TLC detection (developing solvent CH2Cl2:CH3OH=15:1), the solvent was evaporated to dryness using an oil pump. The crude product was separated by silica gel chromatography column and eluted with CH2Cl2:CH3OH=30:1 eluent to obtain the 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 value: 877.3583; Actual value: 877.3582.

[0029] Synthesis of probe IR780T-ER.

[0030] The synthesis method is as follows.

[0031]

[0032] Specific steps: A magnetic magnet, IR780-ER (50 mg, 0.052 mmol), 1,2-bis(4-hydroxybenzene)-1,2-diphenyl ether (75 mg, 0.208 mmol), and potassium carbonate (22 mg, 0.156 mmol) were placed in a 10 mL two-necked flask. A reflux apparatus was assembled, and the mixture was purged three times with vacuum and nitrogen. Then, 2 mL of DMF was added using a syringe. The mixture was stirred at room temperature for 48 h under nitrogen protection. TLC analysis (developing solvent CH2Cl2:CH3OH = 15:1) confirmed complete reaction. The solvent was evaporated to dryness using an oil pump. The crude product was separated using a silica gel column elution with CH2Cl2:CH3OH = 40:1 eluent to obtain the green solid compound IR780T-ER. 1 H 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] + The calculated value is 1205.5279; the actual value is 1205.5283.

[0033] Synthesis of probe T780T-ER.

[0034]

[0035] The synthesis method is as follows.

[0036] Specific steps: The magnetic flux, IR780-ER (20 mg, 0.02 mmol), 1,2-bis(4-hydroxybenzene)-1,2-diphenyl ether (2.53 mg, 0.007 mmol), and potassium carbonate (6 mg, 0.02 mmol) were placed in a 10 mL two-necked flask. A reflux apparatus was assembled, and the mixture was purged three times with vacuum and nitrogen. Then, 2 mL of DMF was added using a syringe. The mixture was stirred at room temperature for 48 h under nitrogen protection. TLC analysis (developing solvent CH2Cl2:CH3OH = 15:1) confirmed complete reaction. The solvent was evaporated to dryness using an oil pump. The crude product was separated using a silica gel column elution with CH2Cl2:CH3OH = 20:1 eluent to obtain the green solid compound T780T-ER. 1 H 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] + The calculated value is 1023.9564; the actual value is 1023.9578.

[0037] Example 2.

[0038] UV-Vis absorption and fluorescence absorption spectra: 10 µM solutions of IR780-ER, IR780T-ER, and T780T-ER in DMSO and aqueous solutions were prepared, and fluorescence and UV absorption spectra were measured. The results are as follows: Figure 2 .

[0039] Example 3.

[0040] ROS generation and photothermal stability: Prepare 2 mL of 5 µM aqueous solutions of IR780-ER, IR780T-ER, and T780T-ER, respectively, and add 10 µM DHR123 solution, 10 µM HPF solution, or 50 µM ABDA solution, respectively, and mix well. Use an 808 nm laser (power 0.3 W / cm²) to... 2 Irradiation was performed for 10 minutes, and the fluorescence spectrum or UV absorption spectrum of the solution was recorded at corresponding time intervals. Fluorescence emission intensity-time at 525 nm (DHR123) and 515 nm (HPF) and UV absorption intensity-time at 378 nm (ABDA) were plotted, respectively. Two mL of 10 µM DMSO solutions of IR780-ER, IR780T-ER, and T780T-ER were prepared and irradiated with 808 nm lasers of different powers (0.3, 0.5, 0.8 W / cm²). 2 Temperature values ​​were recorded every 30 seconds, and temperature changes were recorded from 0 to 10 minutes. Two mL solutions of 10 µMIR780-ER, IR780T-ER, and T780T-ER were prepared and irradiated with an 808 nm laser (power 0.3 W / cm²). 2 The temperature was recorded every 30 seconds for 0-10 minutes. After 10 minutes, the laser was removed, and the solution was allowed to cool naturally. The temperature was recorded again every 30 seconds until the solution returned to its initial temperature. The solution color was then recorded. This process was repeated four times, and a temperature-time curve was plotted. The results are as follows: Figure 3 .

[0041] Example 4.

[0042] Fluorescence confocal laser scanning microscopy imaging: A549 cells were seeded in 35 mm glass-bottomed culture dishes (for confocal microscopy) at a density of 3*102. 5 / plate, incubate overnight. Add probes IR780-ER (5 µM), IR780T-ER (10 µM), and T780T-ER (10 µM) respectively, and incubate for 5 hours. Then add 100 nM ER-tracker Blue or Mito-tracker Green and continue incubation for half an hour. Aspirate the culture medium and wash three times with PBS. Record cell fluorescence using a confocal microscope. Results are as follows. Figure 4 .

[0043] Example 5.

[0044] Cytological morphology and Western blot evaluation of pyroptosis: A549 and 4T1 cells were seeded in 35 mm glass-bottomed culture dishes (for confocal microscopy) at a density of 3 x 10⁻⁶ cells / mL. 5 / plate, incubate overnight. Discard the original medium, add medium containing 5 µM probe T780T-ER, and incubate for 24 h. Laser irradiation group uses 808 nm laser (power 0.3 W / cm²). 2 After irradiation for 5 minutes and subsequent 6-hour culture, cell fluorescence images were observed using a confocal microscope. Western blotting: 4T1 cells were seeded into 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 established by adding culture medium containing 5 µM probe T780T-ER and culturing for 24 hours. The laser irradiation group was irradiated with an 808 nm laser (0.3 W / cm²) for 5 minutes, followed by 6 hours of culture. Cells were then collected and total protein was extracted using lysis buffer. The extracted proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. Non-specific sites were blocked with skim milk (10%). After incubation overnight at 4 °C with diluted primary antibodies (caspase-3, GSDME, β-actin antibody), the PVDF membrane was treated with HRP-bound secondary antibody. Finally, immunoassay readings were obtained using a chemiluminescence system. The results are shown below. Figure 5 .

[0045] Example 6.

[0046] Immunofluorescence confocal laser scanning microscopy imaging of CRT and HMGB1 proteins: 4T1 cells were seeded in 35 mm glass-bottomed culture dishes (for confocal microscopy) at a density of 3*102. 5 / plate, incubate overnight. Discard the original culture medium, set up different groups, add culture medium containing 5 µM probe T780T-ER, and incubate for 24 h. Laser irradiation group is treated with 808 nm laser (power 0.3 W / cm²). 2 After irradiation for 5 minutes and subsequent culturing for 6 hours, cells were fixed with 4% paraformaldehyde for 20 minutes, blocked with 5% BSA for 0.5 hours, and then incubated overnight at 4°C with diluted CRT or HMGB1 antibody (infiltrated with 0.1% Triton-100 for 5 minutes). Finally, cells were incubated with Alexa Fluor 488-labeled secondary antibody for 2 hours and stained with DAPI for 10 minutes. After washing three times with PBS, cell fluorescence images were observed using a confocal microscope. The results are shown below. Figure 6 .

[0047] Example 7.

[0048] In vivo photothermal evaluation and tumor treatment: 1 × 10⁻⁶ cells suspended in 100 μL PBS were used for tumor treatment. 7 4T1 cancer cells were subcutaneously implanted into the right side of each BALB / c mouse as the primary tumor. Five days later, the same number of 4T1 cells were implanted into the left abdomen of the mouse as the distal tumor. When the primary tumor of the mouse reached 100 mm... 3 Mice were then divided into five groups (n = 6). Three groups of mice received intratumoral injection of T780T-ER (100 µM, 100 µL), while the other two groups received PBS (100 µL) as a control group. At 9 and 24 hours, the mice were subjected to 808 nm laser treatment (0.3 W / cm²). 2 Or 0.8 W / cm 2 The primary tumors of two mice in the T780T-ER group and the PBS group were irradiated for 10 minutes, and the tumor temperature was recorded using a thermal imaging camera. The treatment was then repeated twice. Tumor volume was measured every other day and calculated using the following formula: Volume = (Tumor length) × (Tumor width) 2 / 2. After 21 days, all mice were euthanized by cervical dislocation, and major organs were collected for H&E staining. Tumor tissue and spleen from five groups of mice (n=3) were used for FACS analysis, and residual tumor tissue was fixed with 4% formaldehyde solution for IHC and IF analysis.

Claims

1. A near-infrared photosensitizer that targets the endoplasmic reticulum to promote pyroptosis, the structure of which is shown below:

2. The cyanine photosensitizer as described in claim 1, characterized in that: The near-infrared photosensitizer modifies the heptamethrin cyanide molecule, introducing p-toluenesulfonamide and tetraphenylethylene (TPE) groups, which can specifically recognize sulfonamide receptors on the endoplasmic reticulum membrane and specifically target the endoplasmic reticulum, thereby enhancing the photothermal stability and ROS generation efficiency of the photosensitizer.

3. The application of the near-infrared photosensitizer that targets endoplasmic reticulum to promote pyroptosis as described in claim 1 in the preparation of antitumor reagents.

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