AIE photosensitizer capable of inducing pyroptosis as well as preparation method and application of AIE photosensitizer

Abstract

The invention provides an AIE photosensitizer capable of inducing pyroptosis as well as a preparation method and application of the AIE photosensitizer. The preparation method comprises the following steps: carrying out a Knoevenagel condensation reaction between a corresponding aldehyde precursor and 4-pyridyl acetonitrile, and then treating with (4-bromobutyl) triphenyl phosphorus bromide to obtain the AIE photosensitizer capable of inducing pyroptosis. According to the AIE photosensitizer, a cationic side group is incorporated into a molecular structure, so that mitochondrial targeting can be realized; the AIE photosensitizer based on mitochondrial targeting shows powerful and efficient ROS (reactive oxygen species) generation; by generating high-concentration aggregated ROS in mitochondria, the mitochondrial targeting photosensitizer activates a caspase pathway to cause gasdermin lysis, then cells swell and release cell contents, and finally pyroptosis is induced, so that effective TSCC ablation can be realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor treatment, and particularly relates to an AIE photosensitizer capable of inducing cell pyroptosis, and a preparation method and application thereof. Background Art

[0002] Tongue squamous cell carcinoma (TSCC) is the predominant form of tongue cancer, accounting for over 90 out of every 100 tongue cancer cases. Symptoms of TSCC include difficulty eating or swallowing, speech problems, weight loss, and bad breath. This malignancy primarily affects individuals with a familial predisposition to the disease or those who chew betel quid, have vitamin deficiencies, or are infected with the human papillomavirus (HPV). Based on clinical and pathological features, TSCC can be divided into three stages: local, regional, and distant. According to current epidemiological reports, the five-year mortality rate associated with these stages is 68%. Although current treatment options for TSCC include chemotherapy, radiotherapy, and surgical resection, their efficacy remains limited, leading to frequent recurrence and metastasis. Surgical resection is associated with incomplete excision, a high risk of recurrence, and a lack of targeted therapy, which can affect the structure and function of normal tissues. Radiotherapy, as a radiation-dependent treatment, suffers from poor targeting and biosafety, and can easily damage normal cells, thus impacting patients' quality of life. Similar to radiotherapy, chemotherapy lacks targeting and can easily kill cells throughout the body, leading to systemic side effects. Commonly used chemotherapy drugs, such as cisplatin, interfere with cell proliferation by intercalating into DNA structures. However, clinical use can lead to side effects such as neuropathic pain, due to cisplatin's ability to kill normal nerve cells. Therefore, research is urgently needed to develop new TSCC treatments that utilize cutting-edge technologies and innovative materials to improve treatment efficacy and slow disease progression.

[0003] Pyroptosis is a recently elucidated form of programmed cell death characterized by the formation of membrane pores, which induce cell swelling and rupture. This complex process is initiated by the release of calcium ions, mitochondrial damage, caspase cleavage, and subsequent gasdermin activation. Activated caspases then cleave gasdermin, releasing its N-terminal domain, which aggregates and forms gasdermin pores. As a result, cellular contents such as cytokines and lactate dehydrogenase (LDH) are released, triggering a potent antitumor immune response. Compared to apoptosis, pyroptosis shows great potential as a viable avenue for cancer therapy. Unlike apoptosis, which elicits an innate immune response, pyroptosis strongly activates inflammatory responses and is considered a form of immunogenic cell death (ICD). Therefore, pyroptosis could be a powerful tool for the treatment of TSCC. Therefore, the exploration of novel therapeutic agents that can effectively induce pyroptosis in TSCC cells is urgently needed.

[0004] In addition, photodynamic therapy (PDT) is an innovative and promising cancer treatment method with the advantages of precise control and high precision. This treatment method relies on light-activated photosensitizers, which subsequently produce reactive oxygen species (ROS) to induce cell death. However, commercial photosensitizers have many disadvantages, including poor photostability and ACQ (Aggregate-caused quenching) effect when aggregated. In order to overcome these limitations, the use of photosensitizers based on aggregation-induced emission (AIE) to achieve photodynamic therapy has become a promising strategy. AIE-based photosensitizers can produce more ROS in an aggregated state while emitting bright fluorescence, thereby enhancing the production of ROS. Photosensitizers precisely targeted to specific organelles, such as photosensitizers targeting mitochondria, can efficiently produce ROS in important organelles. Summary of the Invention

[0005] In order to overcome the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a class of AIE photosensitizers that can induce cell pyroptosis, as well as a preparation method and application thereof.

[0006] According to a first aspect of the present invention, the present invention provides an AIE photosensitizer that can induce cell pyroptosis, wherein the AIE photosensitizer has the following structure:

[0007]

[0008] According to a second aspect of the present invention, the present invention provides a method for preparing the AIE photosensitizer capable of inducing cell pyroptosis as described above, comprising the following steps:

[0009] Step S1: Add 5-(diphenylamino)thiophene-2-carboxaldehyde, a compound having a structure as shown in Formula 1, and 4-pyridylacetonitrile, as shown in Formula 2, to a solvent of methanol, dissolve them, and then add potassium tert-butoxide to the solution as a catalyst. The reaction mixture is stirred at room temperature, and then the solvent is removed and the residue is purified to obtain a compound 1 as shown in Formula 3.

[0010]

[0011] Step S2: dissolving compound 1 as shown in Formula 3 and (4-bromobutyl)triphenylphosphonium bromide as shown in Formula 4 in 1,2-dichlorobenzene, stirring the mixture to react under nitrogen protection, then cooling the mixture to room temperature and purifying the crude product to obtain the AIE photosensitizer.

[0012] In the preparation method provided by the present invention, in step S1, the mass ratio of 5-(diphenylamino)thiophene-2-carboxaldehyde to 4-pyridylacetonitrile is 1:1 to 1:2; the volume ratio of the mixture of 5-(diphenylamino)thiophene-2-carboxaldehyde and 4-pyridylacetonitrile to methanol is 1:1 to 1:2; the mass ratio of the mixture of 5-(diphenylamino)thiophene-2-carboxaldehyde and 4-pyridylacetonitrile to potassium tert-butoxide is 1:1 to 2:3; and the stirring time is 4h-6h.

[0013] In the preparation method provided by the present invention, in step S2, the mass ratio of compound 1 to (4-bromobutyl)triphenylphosphine bromide is 1:1 to 1:2; the volume ratio of the mixture of compound 1 and (4-bromobutyl)triphenylphosphine bromide to 1,2-dichlorobenzene is 1:1 to 1:2; the reaction temperature is 120-140°C; and the reaction time is 16h-24h.

[0014] According to the third aspect of the present invention, the present invention provides a use of the AIE photosensitizer that can induce cell pyroptosis as described above in the preparation of a drug for triggering tumor cell pyroptosis and a drug component for triggering tumor cell pyroptosis.

[0015] In the application provided by the present invention, the tumor cells are tongue squamous cell carcinoma cells.

[0016] According to a fourth aspect of the present invention, the present invention provides a use of the AIE photosensitizer capable of inducing cell pyroptosis as described above in the preparation of a photodynamic agent for tumor treatment.

[0017] The implementation of the present invention can achieve the following beneficial effects: the present invention obtains an AIE photosensitizer that can induce cell pyroptosis through a Knoevenagel condensation reaction between the corresponding aldehyde precursor and 4-pyridylacetonitrile, followed by treatment with (4-bromobutyl)triphenylphosphonium bromide; the AIE photosensitizer incorporates cationic side groups into the molecular structure, thereby achieving mitochondrial targeting; the mitochondrial-targeted AIE photosensitizer exhibits strong and efficient ROS generation; by generating high concentrations of aggregated ROS in the mitochondria, the mitochondrial-targeted photosensitizer activates the caspase pathway, leading to gasdermin cleavage, followed by cell swelling and release of cell contents, and ultimately inducing cell pyroptosis, thereby achieving effective TSCC ablation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only embodiments of the present invention. Those skilled in the art can also derive other drawings based on the provided drawings without inventive work.

[0019] Figure 1 Shown is the NMR spectrum of the intermediate product Compound 1 prepared in Example 1;

[0020] Figure 2 Shown is the NMR spectrum of the AIE photosensitizer Th-M that can induce cell pyroptosis prepared in Example 1;

[0021] Figure 3 Shown are high-resolution mass spectra of the intermediate product compound 1 and the AIE photosensitizer prepared in Example 1;

[0022] Figure 4 Shown is the AIE curve of the AIE photosensitizer Th-M prepared in Example 1;

[0023] Figure 5 Shown is the detection curve of ROS generated by the AIE photosensitizer Th-M prepared in Example 1;

[0024] Figure 6 Shown are the mitochondrial imaging images of the AIE photosensitizer Th-M prepared in Example 1 and the fitting graph and Pearson coefficient of the commercial mitochondrial probe;

[0025] Figure 7 Shown is the therapeutic effect of the AIE photosensitizer Th-M prepared in Example 1 on tongue cancer;

[0026] Figure 8 Shown are the GSDME expression test results of the AIE photosensitizer prepared in Example 1. DETAILED DESCRIPTION

[0027] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments of the present invention are now described in detail with reference to the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, and not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments described in this specification without inventive effort are also within the scope of protection of the present invention.

[0028] The present invention provides a method for preparing an AIE photosensitizer that can induce cell pyroptosis. The synthesis route is as follows:

[0029]

[0030] The preparation method comprises the following steps:

[0031] Step S1: Add 5-(diphenylamino)thiophene-2-carboxaldehyde, a compound having a structure as shown in Formula 1, and 4-pyridylacetonitrile, as shown in Formula 2, to a methanol solvent, dissolve the mixture, and then add potassium tert-butoxide as a catalyst to the solution. The reaction mixture is stirred at room temperature, and then the solvent is removed and the residue is purified to obtain a compound 1 as shown in Formula 3:

[0032]

[0033] Specifically, in one embodiment of the present invention, 5-(diphenylamino)thiophene-2-carboxaldehyde and 4-pyridylacetonitrile are first added to a round-bottom flask at a mass ratio of 1:1 to 1:2. Then, methanol is added as a solvent for dissolution, wherein the volume ratio of the mixture of 5-(diphenylamino)thiophene-2-carboxaldehyde and 4-pyridylacetonitrile to methanol is 1:1 to 1:2. After dissolution, potassium tert-butoxide is added as a catalyst and stirred at room temperature for 4-6 hours to allow a Knoevenagel condensation reaction to occur between 5-(diphenylamino)thiophene-2-carboxaldehyde and 4-pyridylacetonitrile, wherein the mass ratio of the mixture of 5-(diphenylamino)thiophene-2-carboxaldehyde and 4-pyridylacetonitrile to potassium tert-butoxide is 1:1 to 2:3. A lower or higher raw material ratio may lead to an increase in side reactions, affecting the purity and yield of the target product. Then, the solvent is removed under pressure. The method for removing methanol is preferably to remove methanol by distillation under reduced pressure. The parameters are not particularly limited. A distillation method for removing methanol under reduced pressure well known in the art can be used. After removing the methanol, the residue is purified to obtain Compound 1. The purification method is preferably silica gel chromatography purification. The parameters are not particularly limited. A silica gel chromatography purification method well known in the art can be used.

[0034] Step S2: dissolving compound 1 as shown in Formula 3 and (4-bromobutyl)triphenylphosphonium bromide as shown in Formula 4 in 1,2-dichlorobenzene, stirring the mixture to react under nitrogen protection, then cooling the mixture to room temperature and purifying the crude product to obtain the AIE photosensitizer.

[0035]

[0036] Specifically, in one embodiment of the present invention, the obtained compound 1 is mixed with triphenylphosphine bromide in a mass ratio of 1:1 to 1:2; then, a solvent 1,2-dichlorobenzene is added for dissolution, wherein the volume ratio of the mixture of compound 1 and (4-bromobutyl)triphenylphosphine bromide to 1,2-dichlorobenzene is 1:1 to 1:2; a lower or higher raw material ratio may lead to an increase in side reactions, affecting the purity and yield of the target product. After dissolution, the reaction is carried out at a temperature of 120-140°C under nitrogen protection for 16h-24h. Then, the mixture is cooled to room temperature, and the crude product is purified to obtain the photosensitizer. The purification method is preferably silica gel chromatography purification. The parameters are not particularly limited, and the silica gel chromatography purification method well known in the art can be used.

[0037] Example 1 Synthesis of Th-M

[0038] (1) 5-(Diphenylamino)thiophene-2-carboxaldehyde (559 mg, 2 mmol) and 4-pyridylacetonitrile (236 mg, 2 mmol) were added to a round-bottom flask and dissolved in 10 mL of methanol. After dissolution, potassium tert-butoxide (336 mg, 3 mmol) was added. The reaction mixture was stirred at room temperature for 5 hours. Then, the solvent was removed under pressure. The residue was purified to obtain compound 1. Its NMR data are as follows:

[0039] 1H NMR (500MHz, CDCl3): δ8.59 (s, 2H), 7.68 (s, 1H), 7.43 (d, J = 3.1Hz, 3H), 7.37 (d,J=6.2Hz,4H),7.28(d,J=7.0Hz,4H),7.22(d,J=5.7Hz,2H),6.46(s,1H).

[0040] (2) The obtained compound 1 (380 mg, 1 mmol) and (4-bromobutyl)triphenylphosphine bromide (478 mg, 1 mmol) were dissolved in 1,2-dichlorobenzene (10 mL) and filled with nitrogen. The mixture was stirred at 130°C overnight. After the mixture was cooled to room temperature, the crude product was purified to obtain Th-M. Its NMR data were:

[0041] 1H NMR (500MHz, MeOD): δ8.65(d,J=6.9Hz,2H),8.54(s,1H),7.95(d,J=7.0Hz,2H ),7.93-7.83(m,10H),7.78(td,J=7.9,3.5Hz,6H),7.53(t,J=7.8Hz,4H),7.46 (d,J=7.6Hz,4H),7.42(s,2H),6.48(d,J=4.6Hz,1H),4.53(t,J=7.4Hz,2H),3 .60(t,J=15.0Hz,2H),2.32-2.23(m,2H),1.81-1.71(m,2H).HRMS:calculated for[C 46 H 40 N3P2S] 2+ :348.6335; found:345.6340.

[0042] Example 2 Photophysical properties of Th-M

[0043] First, the AIE curve of Th-M was tested. Th-M can be well dissolved in dimethyl sulfoxide, but its solubility in toluene is poor. Therefore, we chose dimethyl sulfoxide and toluene as the good solvent and poor solvent for the AIE curve test. First, Th-M was dissolved in DMSO, and then toluene was added at 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 95% and the final concentration was kept at 10 -5 M, and then use a fluorescence spectrometer to test its photoluminescence spectrum under 550nm excitation and record it. The fluorescence intensity value of the maximum emission wavelength is I divided by the fluorescence intensity value of Th-M when the toluene content is 0% I0 as the vertical axis. Figure 4 As shown in Figure 3, with the increase of toluene content, the luminescence intensity of Th-M begins to increase, showing obvious AIE characteristics.

[0044] Example 3 ROS production ability test of Th-M

[0045] Th-M has a clear donor-acceptor structure, suggesting that it can generate ROS. In order to verify whether it generates ROS, DCFH-DA was used as a total ROS indicator. If Th-M generates ROS under white light irradiation, DCFH-DA will emit green fluorescence under excitation. Figure 5 As shown, compared with the control group, the fluorescence of the group added with 10 micromolar Th-M was significantly enhanced after white light irradiation, which suggests that Th-M can produce ROS under light.

[0046] This result confirms that Th-M does have the ability to generate ROS, which is consistent with its donor-acceptor structure. ROS, as intracellular oxidative stress molecules, play an important role in tumor treatment, especially in photodynamic therapy. The ROS produced by Th-M may help to exert a destructive effect on tumor cells, thereby achieving the effect of photodynamic therapy. This experimental result provides an important experimental basis for further research on the application of Th-M in photodynamic therapy and tumor treatment. The ROS-generating ability of Th-M supports its potential as a photosensitizer in tumor treatment and provides inspiration for future research and clinical application directions.

[0047] Example 4 Mitochondrial colocalization test of Th-M

[0048] Th-M has a triphenylphosphine structure at one end, which suggests that Th-M has targeting properties to mitochondria in cells. Therefore, 1 micromolar commercial mitochondrial probe and 5 micromolar Th-M were added to the cell culture dish and stained for 30 minutes. The luminescence of the cells in the culture dish was observed under a confocal microscope. Figure 6 As shown, we found that the commercial mitochondrial probe and Th-M co-stained mitochondria well with a Pearson coefficient of 0.91.

[0049] The results of this study indicate that Th-M does have the ability to target mitochondria. This experimental result not only verifies the targeting of Th-M to mitochondria, but also demonstrates its potential in cell mitochondrial imaging. Through staining experiments, Th-M showed good co-localization with commercial mitochondrial probes, which provides strong support for its future cell imaging research and application. In addition, due to the targeting of Th-M within mitochondria and its ability to produce ROS, this photosensitizer is of great significance for triggering cell apoptosis through mitochondria in photodynamic therapy. The results of this study have laid a solid foundation for the application of Th-M in fields such as photodynamic therapy and cell imaging, and provide new ideas for the development of more targeted and effective photosensitizers.

[0050] Example 5 Anti-tumor test of Th-M

[0051] Th-M targets mitochondria and can produce ROS, so we tested its anti-tumor ability. First, we established a nude mouse tumor growth model using the oral epithelial cancer cell line CAL27. Second, we injected Th-M directly into the mouse tumor, waited for 24 hours, and then irradiated the mouse with 50 milliwatts per square centimeter of white light and recorded the tumor size. Figure 7 As shown, the simultaneous injection of Th-M and white light irradiation group produced a significant tumor elimination effect, and the Th-M injection group did not show significant body weight changes.

[0052] The experimental results showed that direct injection of Th-M into mouse tumors and subsequent irradiation with 50 milliwatts per square centimeter of white light one day later resulted in significant tumor elimination, demonstrating that Th-M has excellent photodynamic therapeutic efficacy. This result provides strong support for the application of Th-M in tumor treatment. Furthermore, the experimental results also showed that the mice in the Th-M injection group did not experience significant weight changes, indicating the high biosafety and reliability of Th-M. This result provides a preliminary assessment of the safety and tolerability of Th-M as a potential tumor treatment drug, providing a reference for its future clinical application. The validation of this experimental result can further promote the research and development of Th-M in the field of tumor treatment and provide an important experimental basis for the development of more effective tumor treatment drugs.

[0053] Example 6 Th-M cell pyroptosis test

[0054] Th-M targets mitochondria and can effectively kill tumor cells. Therefore, after observing its death morphology, we inferred that its mode of death was pyroptosis. Pyroptosis is a specific form of cell apoptosis, and its mechanism is related to the intracellular stomatal protein gasdermin. Therefore, we used immunoblotting technology to test the expression of pyroptosis-related proteins before tumor cell death. At the same time, the expression of apoptosis-related proteins was also tested. Figure 8 As shown, GSDME had no cleavage effect in the control group, the light-only group, and the Th-M-only incubation group, and GSDME cleavage was only detected six hours after light exposure. This result suggests that the killing effect of Th-M on tumors is achieved through cell pyroptosis.

[0055] Immunoblotting confirmed the effects of Th-M in tumor cells and its potential to trigger pyroptosis in tumor cells. Th-M activates the caspase pathway, leading to the cleavage of gasdermin, ultimately causing cell swelling and the release of cellular contents, thereby inducing pyroptosis. This tumor cell-specific mechanism offers new insights and potential for the application of Th-M in tumor therapy.

[0056] The images taken with a confocal microscope showed the changes in cells after treatment with an AIE-based photosensitizer, confirming that the cells swelled. In addition, Th-M verified the cleavage of gasderminE by Western blotting under white light irradiation. Subsequently, the anti-tumor effect of Th-M in the human tongue squamous cell carcinoma cell line (CAL27) under white light irradiation was very significant. At the same time, in order to understand the therapeutic effect of Th-M, the present invention conducted experiments using the CAL27 nude mouse tumor model. It is worth noting that the results showed that Th-M has an excellent therapeutic effect, which proves that it has the potential to be used as an effective treatment strategy for tongue cancer through cell charring.

[0057] The AIE photosensitizer Th-M provided by the present invention is a fluorescent active molecule with a donor-acceptor structure and a triphenylphosphine structure. This structure enables Th-M to exhibit strong and efficient ROS generation ability and mitochondrial targeting. ROS is an important cellular oxidative stress molecule and plays a vital role in photodynamic therapy. Th-M has a good photodynamic therapeutic effect in tumor treatment by targeting mitochondria and producing high concentrations of aggregated ROS in mitochondria. At the same time, the expression of pyroptosis-related proteins before tumor cell death was tested by immunoblotting technology, which confirmed that it can activate the caspase pathway, leading to gasdermin cleavage, followed by cell swelling and release of cell contents, and ultimately inducing cell pyroptosis. Therefore, it has the potential to prepare drugs for triggering tumor cell pyroptosis and drug components for triggering tumor cell pyroptosis. Therefore, as a potential tumor treatment drug, Th-M has the potential to develop in the direction of photodynamic therapy and triggering cell pyroptosis, providing important reference and inspiration for future tumor treatment research.

[0058] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific embodiments. The above-mentioned specific embodiments are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can make many modifications without departing from the scope of protection of the present invention and the claims, and all of these belong to the scope of protection of the present invention.

Claims

1. An AIE photosensitizer capable of inducing cell pyroptosis, characterized in that: The AIE photosensitizer has the structure shown below:

2. A method for preparing an AIE photosensitizer capable of inducing cell pyroptosis according to claim 1, characterized in that: The following steps are involved: Step S1: Add 5-(diphenylamino)thiophene-2-carboxaldehyde, a compound having a structure as shown in Formula 1, and 4-pyridylacetonitrile, as shown in Formula 2, to a solvent of methanol, dissolve them, and then add potassium tert-butoxide to the solution as a catalyst. The reaction mixture is stirred at room temperature, and then the solvent is removed and the residue is purified to obtain a compound 1 as shown in Formula 3. Step S2: dissolving compound 1 as shown in Formula 3 and (4-bromobutyl)triphenylphosphonium bromide as shown in Formula 4 in 1,2-dichlorobenzene, stirring the mixture to react under nitrogen protection, then cooling the mixture to room temperature and purifying the crude product to obtain the AIE photosensitizer.

3. The preparation method according to claim 2, wherein In step S1, the mass ratio of 5-(diphenylamino)thiophene-2-carboxaldehyde to 4-pyridylacetonitrile is 1:1 to 1:2; the volume ratio of the mixture of 5-(diphenylamino)thiophene-2-carboxaldehyde and 4-pyridylacetonitrile to methanol is 1:1 to 1:2; the mass ratio of the mixture of 5-(diphenylamino)thiophene-2-carboxaldehyde and 4-pyridylacetonitrile to potassium tert-butoxide is 1:1 to 2:3; and the stirring time is 4h-6h.

4. The preparation method according to claim 2, wherein In step S2, the mass ratio of compound 1 to (4-bromobutyl)triphenylphosphonium bromide is 1:1 to 1:2; the volume ratio of the mixture of compound 1 and (4-bromobutyl)triphenylphosphonium bromide to 1,2-dichlorobenzene is 1:1 to 1:2; the reaction temperature is 120-140° C.; and the reaction time is 16-24 hours.

5. Use of the AIE photosensitizer capable of inducing cell pyroptosis as claimed in claim 1 in the preparation of a drug for triggering tumor cell pyroptosis and a drug component for triggering tumor cell pyroptosis.

6. The use according to claim 5, characterized in that The tumor cells are tongue squamous cell carcinoma cells.

7. Use of the AIE photosensitizer capable of inducing cell pyroptosis as claimed in claim 1 in the preparation of a photodynamic agent for tumor treatment.

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