Preparation method and application of heptamethine cyanine dye with controllable pyroptosis activity

Abstract

The invention discloses a heptamethine cyanine photosensitizer with controllable pyroptosis intensity as well as a preparation method and application of the heptamethine cyanine photosensitizer. The method comprises the following steps: by taking IR808 as a parent nucleus, firstly, introducing tetraphenylethylene (TPE) into a meso-position to remarkably improve intersystem crossing efficiency, and converting the IR808 with negative pyroptosis into T808 with positive pyroptosis; then, PEG chains with the molecular weights of 550 Da, 1000 Da and 200 Da are connected to carboxyl on one side of the T808, and PT550, PT1000 and PT2000 are obtained. Along with the extension of a PEG chain, the hydrophilicity of the molecule is gradually enhanced, and the intensity of inducing the pyroptosis of tumor cells under the irradiation of 808 nm near-infrared light is PT550gt in sequence; t808gt, T808gt; pT1000 < gt >; and PT2000, so that accurate gradient regulation and control of pyroptosis activity are realized. The series of photosensitizers have excellent phototherapy performance and can be used for phototherapy of solid tumors.

Description

Technical Field

[0001] This invention belongs to the field of phototherapy technology, specifically a class of photosensitizers with controllable pyrolysis intensity, their preparation methods, and applications. Background Technology

[0002] Phototherapy utilizes photosensitizers to generate reactive oxygen species (ROS) or localized heat under specific wavelengths of light, achieving precise killing of tumor cells. It offers significant advantages such as spatiotemporal control, non-invasiveness, and low toxicity. Pyroptosis is a highly inflammatory programmed cell death process. It occurs when the N-terminal fragment (GSDM-N) of gasdermin family proteins oligomerizes on the cell membrane, forming pores. This leads to cell lysis and the release of large amounts of pro-inflammatory factors such as IL-1β and IL-18, inducing strong immunogenic cell death (ICD) and activating the body's anti-tumor immune response. Combining phototherapy with pyroptosis is considered an ideal strategy to enhance the efficacy of tumor immunotherapy.

[0003] Heptamethrin dyes (such as IR808 and IR780) are a class of classic near-infrared photosensitizers, possessing excellent tissue penetration depth and easily modifiable structures. However, their parent molecules themselves have extremely weak or negative pyroptosis induction capabilities, and pyroptosis activity is influenced by multiple factors, including molecular structure, subcellular localization, and aggregation state, resulting in a complex regulatory mechanism. Currently, most pyroptosis-inducing photosensitizers rely on accidental discovery or simple splicing of subcellular organelle targeting groups, lacking systematic and predictable rational design strategies. Furthermore, the intensity of pyroptosis is difficult to precisely control; excessive pyroptosis can even trigger harmful systemic inflammatory storms. PEGylation is a common strategy for regulating the hydrophilicity / hydrophobicity, aggregation behavior, and subcellular distribution of photosensitizers, but current research mainly focuses on pharmacokinetic optimization, and no studies have explored the dose-response relationship between PEG chain length and pyroptosis induction intensity. Summary of the Invention

[0004] The purpose of this invention is to provide a class of photosensitizers with controllable pyrolysis intensity, their preparation methods, and applications.

[0005] To achieve the above objectives, this invention utilizes a two-step rational design to realize the transformation of heptamethrin-based photosensitizers from pyroptosis-negative to pyroptosis-positive, and then to a pyroptosis intensity gradient, specifically including: 1) A strong electron-donating tetraphenylethylene (TPE) unit was introduced into the mesonephrine core (IR808) to obtain compound T808. Theoretical calculations and photophysical tests confirmed that this modification significantly promoted intersystem crossing (ISC), converting the pyroptosis-negative parent compound IR808 into the pyroptosis-positive photosensitizer T808; 2) Based on the aforementioned pyrolysis positive molecule T808, PEG chains of different molecular weights (550, 1000, 2000 Da) were further linked to obtain compounds PT550, PT1000, and PT2000, respectively. The modification of the polyethylene glycol chain caused the pyrolysis intensity to follow the pattern of PT550 > T808 without PEG > PT1000 > PT2000.

[0006] The pyrolysis-inducible photosensitizer provided by this invention has the following general structural formulas (I) and (II):

[0007] Formula (I) represents compounds of the T808 class, and Formula (II) represents specific structural formulas of compounds of the PT series.

[0008] The chemical synthesis route for preparing the pyroptosis molecule proposed in this invention is as follows:

[0009] The specific steps of preparation are as follows: (1) Synthesis of T808 Under nitrogen protection, using anhydrous N,N-dimethylformamide as solvent, IR808, 4-(1,2,2-triphenylvinyl)phenol, and potassium carbonate were dissolved in an ice bath and stirred for 0.5 hours. The mixture was then brought to room temperature and stirred in the dark for 12 hours, and the solvent was evaporated to dryness. The crude product was purified by silica gel column chromatography to obtain T808.

[0010] (2) Synthesis of PT series compounds (PT550, PT1000, PT2000) Under nitrogen protection, T808 and N,N-diisopropylethylamine were first dissolved in anhydrous N,N-dimethylformamide and stirred for 30 minutes. HBTU and mPEG-NH2 of different molecular weights (550; 1000; 2000) were dissolved in anhydrous N,N-dimethylformamide (1 mL) and added dropwise to the reaction system at 4 °C. The mixture was stirred overnight at room temperature in the dark. The crude product was purified by thin-layer chromatography to obtain the PT series compounds.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0012] The small molecule fluorescent probe of heptamethrin of the present invention has the following characteristics: (1) The fluorescent probe of the present invention introduces tetraphenylethylene molecules on the chlorine atom of cyclohexene in heptamethrin molecules to increase the intersystem crossing rate (ISC) of the molecules, so that the pyroptosis-negative IR808 molecules are transformed into pyroptosis-positive T808 molecules; (2) The fluorescent probe of the present invention introduces polyethylene glycol side chains of different lengths into the pyroptosis-positive T808 to regulate the hydrophilicity and hydrophobicity, aggregation and organelle targeting behavior of the molecules, thereby achieving controllable pyroptosis; (3) The present invention provides clear molecular design guidelines, using TPE to increase ISC to turn on the pyroptosis switch, and then using a specific PEG chain length to achieve intensity fine-tuning. Attached Figure Description

[0013] Figure 1 The images show the UV absorption and fluorescence emission of the photosensitizer (10 µM) of this invention in DMSO and aqueous solution, respectively.

[0014] Figure 2 This is a bar chart showing the Log P values ​​of the photosensitizers T808, PT550, PT1000, and PT2000 of this invention.

[0015] Figure 3 The diagram shows a comparison of type I and type II ROS generation and a photothermal curve for the photosensitizer of this invention.

[0016] Figure 4 This is a confocal microscopy image showing the co-localization of the photosensitizer of this invention with Mito-Tracker Green in 4T1 cells.

[0017] Figure 5 The image shows the cell morphology of pyroptosis induced in 4T1 cells by light exposure using the photosensitizers T808, PT550, P1000, and PT2000 of this invention.

[0018] Figure 6 The photosensitizers T808, PT550, P1000 and PT2000 of this invention induced pyroptosis-related protein immunoblotting in 4T1 cells after light exposure.

[0019] Figure 7 Photothermal images and therapeutic effects of the photosensitizers T808, PT550, and P1000 of this invention in 4T1 tumor-bearing mice. Detailed Implementation

[0020] 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.

[0021] The following description, in conjunction with the accompanying drawings and examples, further illustrates the specific implementation of the fluorescent probe of the present invention, its preparation method, and its application.

[0022] Example 1:

[0023] Synthesis of T808:

[0025] The specific synthesis route is as follows:

[0026] The synthesis method is as follows: Specific steps: Under nitrogen protection, IR808 (35.06 mg, 0.046 mmol), 4-(1,2,2-triphenylvinyl)phenol (24.02 mg, 0.069 mmol), and potassium carbonate (25.00 mg, 0.18 mmol) were dissolved in anhydrous N,N-dimethylformamide (2 mL), and stirred in an ice bath for 0.5 hours. The reaction mixture was then stirred at room temperature in the dark for 12 hours. The reaction solution was diluted with dichloromethane, washed with saturated sodium chloride solution, and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 10:1, v / v elution) to finally obtain green solid T808 (30.08 mg, 0.028 mmol, yield 61%). 1 H NMR (400 MHz, Methanol- d 4 ) δ 7.93 (d, J = 14.1 Hz, 2H, CH),7.46 - 6.87 (m, 27H, ArH), 6.14 (d, J = 14.2 Hz, 2H, CH), 4.10 (t, J = 7.5 Hz, 4H, NCH2), 2.70 (t, J = 6.0 Hz, 4H, CH2CH), 2.24 (t, J= 7.3 Hz, 4H, CH2CO), 2.00(s, 2H, CH2), 1.81 (m, 4H, CH2), 1.69 (m, 4H, CH2), 1.48 (m, 4H, CH2), 1.39(s, 12H, CH3). 13 C NMR (101 MHz, Methanol- d 4 ) δ 172.18, 162.11, 143.62, 142.22,141.84, 141.11, 138.39, 130.84, 127.56, 127.35, 124.89, 122.01, 121.87,113.75, 110.19, 99.66, 48.92, 143.44, 33.47, 27.16, 26.60, 25.98, 24.34,23.16. HRMS (ESI): m / z 995.5367 ([M-Br] + , calcd, 995.5357).

[0027] Example 2: Preparation of polyethylene glycol-modified heptamethine cyanide molecules PT550, PT1000, and PT2000; the compound structural formulas are as follows:

[0028] The specific synthesis route is as follows:

[0029] The synthesis method is as follows: Specific steps: Under nitrogen protection, T808 (21.49 mg, 0.020 mmol) and N,N-diisopropylethylamine (7.75 mg, 0.060 mmol) were dissolved in anhydrous N,N-dimethylformamide (1 mL) and stirred for 30 minutes. Then, HBTU (30.34 mg, 0.080 mmol) and mPEG-NH2 of different molecular weights (molecular weight 550: 24.20 mg, 0.044 mmol; molecular weight 1000: 44.00 mg, 0.044 mmol; molecular weight 2000: 88.00 mg, 0.044 mmol) were dissolved in anhydrous N,N-dimethylformamide (1 mL) and added dropwise to the reaction system at 4 °C. The mixture was stirred overnight at room temperature in the dark. The reaction mixture was purified by thin-layer chromatography (dichloromethane:methanol = 25:1, v / v) to obtain green solid polyethylene glycol molecules (PT550: 19.00 mg, 0.0086 mmol, yield 43%; PT1000: 20.99 mg, 0.0072 mmol, yield 36%; PT2000: 47.73 mg, 0.0094 mmol, yield 47%). Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) confirmed the molecular weights of the PT series compounds: PT550: expected molecular weight approximately 2095; actual molecular weight approximately 2210; PT1000: expected molecular weight approximately 2959; actual molecular weight approximately 2915; PT2000: expected molecular weight approximately 4976; actual molecular weight approximately 5078.

[0030] Example 3: 10 µM solutions of T808, PT550, PT1000, and PT2000 in DMSO and PBS were prepared respectively, and the UV absorption spectra and fluorescence emission spectra were measured. The results are as follows: Figure 1 .

[0031] Example 4: The photosensitizer was dissolved at 20 μM in a mixture of aqueous and oil phases. After incubation at 37 °C in the dark for 2 h, the mixture was centrifuged to separate the phases. Samples from both the oil and aqueous phases were diluted with DMSO and sonicated to ensure complete dissolution of the photosensitizer. The UV-vis absorbance was then measured, and the concentration of the photosensitizer in both phases was calculated based on the calibration curve. The Log P value was then calculated, and the results are as follows: Figure 2 .

[0032] Example 5: Prepare 2 mL of 5 µM aqueous solutions of T808, PT550, PT1000, and PT2000 respectively, and add 10 µM DCFH-DA or 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 Irradiate for 10 minutes, and record the fluorescence spectrum or UV absorption spectrum of the solution at corresponding time intervals. Plot the fluorescence emission intensity-time at 525 nm (DCFH-DA, DHR123) and 515 nm (HPF), and the UV absorption intensity-time at 378 nm (ABDA), respectively. Prepare 10 µM DMSO solutions of T808, PT550, PT1000, and PT2000, and 2 mL of aqueous solutions, respectively, and use an 808 nm laser (power 0.3 W / cm²). 2 Irradiate for 10 minutes, record the temperature every 30 seconds, and record the temperature change from 0 to 10 minutes. The results are as follows: Figure 3 .

[0033] Example 6: Mitochondrial confocal fluorescence scanning microscopy imaging: 4T1 cells were seeded in 35 mm glass-bottomed culture dishes (for confocal microscopy) at a density of 4*102. 5 / plate, incubate overnight. Add 2.5 µM T808, PT550, PT1000 and PT2000 respectively and incubate for 6 hours, then add Mito-tracker Green and continue incubation for half an hour. Aspirate the culture medium and wash 3 times with PBS. Record cell fluorescence using a confocal microscope. Results are shown below. Figure 4 .

[0034] Example 7: Cytological morphology and Western blot evaluation of pyroptosis: 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 medium and add medium containing different concentrations (2.5 µM, 5 µM, 8 µM, 10 µM) of probes T808, PT550, PT1000, and PT2000, and incubate for 6 h. Laser irradiation group was treated with an 808 nm laser (power 0.3 W / cm²). 2 After irradiating for 5 minutes and culturing for 2 hours, the cell fluorescence images were observed using a confocal microscope. The results are as follows. Figure 5 .

[0035] Example 8: Western blot analysis: 4T1 cells were seeded into 6-well plates at a density of 200,000 cells / well and cultured for 24 h. The original culture medium was discarded, and different groups were established by adding culture medium containing 2.5 µM probes T808, PT550, PT1000, and PT2000, and culturing for 6 h. The laser irradiation group was treated with an 808 nm laser (power 0.3 W / cm²). 2 After irradiation for 5 minutes and incubation for 2 hours, cells were 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%). The membrane was incubated overnight at 4 °C with diluted primary antibody (caspase-3, GSDME, β-actin antibody), followed by treatment with HRP-bound secondary antibody. Finally, an immunoassay was performed using a chemiluminescence system. The results are shown below. Figure 6 .

[0036] Example 9: 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 dorsal side of each BALB / c mouse, and the tumor volume was increased to 180 mm. 3 The mice were then divided into eight groups (n = 6). Four of these groups received intratumoral injections of PBS, T808, PT550, and PT1000 (100 µM, 100 µL), followed by intratumoral injections of an 808 nm laser (0.3 W / cm²) at 2 and 24 hours later. 2 Or 0.8 W / cm 2 The tumors were irradiated for 10 minutes, and the tumor temperature was recorded using a thermal imaging camera. The other four groups received intratumoral injections of PBS, T808, PT550, and PT1000 (100 µM, 100 µL), without light treatment. Treatment was repeated four times. Tumor volume was measured every other day and calculated using the following formula: Volume = (Tumor length) × (Tumor width) 2 / 2. Fourteen days after treatment, all mice were euthanized by cervical dislocation, and major organs were collected for H&E staining. Tumor tissue was fixed with 4% formaldehyde solution for IHC and IF analysis. Experimental results are as follows: Figure 7 .

Claims

1. A heptamethine cyanine compound having controllable pyroptosis activity, characterized in that, The compound is compound T808 or a compound having general formula (II):

2. wherein, The average molecular weight of mPEG is 550 Da, 1000 Da or 2000 Da, respectively.

3. The septamethine indocyanine compound according to claim 1, characterized in that, The compound is selected from one of the following: (1) compound T808; (2) compound PT550 with an average molecular weight of mPEG of 550 Da; (3) compound PT1000 with an average molecular weight of mPEG of 1000 Da; (4) compound PT2000 with an average molecular weight of mPEG of 2000 Da. 4.The heptamethine cyanine compound of claim 2, wherein The n-octanol / water partition coefficient (Log P, 37 °C, shake flask method) of T808, PT550, PT1000 and PT2000 is 1.29 ± 0.10, 0.91 ± 0.10, -0.35 ± 0.10 and -1.16 ± 0.10, respectively.

5. The septamethine chloride compound of claim 2 can affect the ability of organelle targeting by regulating the length of PEG chain, and the Pearson correlation coefficient of co-localization with mitochondrial dye Mito-Tracker Green is about 0.81 for T808, about 0.90 for PT550, about 0.91 for PT1000, and about 0.69 for PT2000, respectively.

6. The septamethine indocyanine compound according to claim 2, characterized in that, Under 808 nm near-infrared light irradiation, the pyroptosis induction intensity of the series of compounds at a concentration of 2.5 μM is in the order of PT550 > T808 > PT1000 > PT2000.

7. Use of the septamethine chloride compound of any one of claim 2 in the preparation of a medicament for the photodynamic treatment of tumors.

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