A novel aie photosensitizer and a preparation method and application thereof

Abstract

The application discloses a novel AIE photosensitizer and a preparation method and application thereof. The AIE photosensitizer of the application comprises TDQ shown in the following structural formula, has superior photo-thermal conversion performance and photodynamic performance, and can be used for preparing a tumor diagnosis reagent or a tumor treatment drug. The co-loading liposome of TDQ and a senescence-inducing molecule (Ali) can be used as a multi-modal light diagnosis and treatment agent to realize efficient delivery of drugs to tumor tissues, and the photo-thermal therapy and the photodynamic therapy based on TDQ not only induce cell apoptosis through oxidative stress and mitochondrial dysfunction, but also trigger cancer cell senescence and promote the secretion of SASPs, so that the anti-tumor immunity is significantly enhanced, and finally the growth of primary tumors, tumor recurrence and re-metastasis are inhibited. The material of the application has simple preparation process, low cost, and is suitable for scale production and clinical medical use.

Description

Technical Field

[0001] This invention belongs to the field of tumor treatment, specifically relating to a novel AIE photosensitizer, its preparation method, and its application in the preparation of tumor treatment drugs. Background Technology

[0002] Recently, immunotherapy for breast cancer has been continuously improved and has become one of the most promising strategies for treating refractory cases. Although immunotherapy can stimulate innate and adaptive immunity to suppress tumor progression, the immunosuppressive environment in the tumor microenvironment (TME) remains a persistent obstacle. Current approaches to remodeling the TME include immune checkpoint inhibitors, cancer vaccines, and induced immunogenic cell death (ICD). ICDs drive the recruitment of antigen-presenting cells (APCs) and the activation of cytotoxic T cells by releasing tumor-associated antigens, damage-associated molecular patterns (DAMPs), and inflammatory factors. However, in highly immunosuppressive and hypoxic cancers (such as triple-negative breast cancer (TNBC)), the conditions of the TME may attenuate the activity of photosensitizers and the induction of antitumor immunity.

[0003] Treatment-induced senescence (TIS) has recently emerged as a complementary strategy, enhancing immunotherapy while suppressing tumor growth. Senescence is a permanent cell cycle arrest triggered by stress, releasing various senescence-associated secretory phenotype (SASP) factors that recruit immune cells to clear senescent tumor cells. Clinically, drugs such as CDK4 / 6 inhibitors and Aurora kinase inhibitors have been shown to induce irreversible cell cycle arrest and immune-mediated tumor clearance through SASPs. Senescence-associated antigen presentation synergistically with immunogenic SASPs can recruit and activate immune cells, thereby promoting adaptive immunity and delaying tumor growth. However, inappropriate dosages or the use of these drugs alone can produce immunosuppressive SASPs, which may promote tumor resistance and recurrence. Therefore, finely modulating SASP output in TIS to maintain its immunostimulatory function while minimizing its pro-tumor effects remains a key challenge for senescence-based therapeutic strategies. Current research suggests that combining senescence-inducing drugs with additional therapeutic modalities can enhance immune activation and therapeutic efficacy.

[0004] Phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), has become a powerful means of inducing ICD by modulating the generation of reactive oxygen species (ROS) and photothermal effects with high spatiotemporal controllability and biocompatibility. Photosensitizers based on organic fluorescent materials have been widely developed due to their promising prospects in image-guided therapy. However, the planar structure of traditional fluorescent materials often leads to π-π stacking conformations, resulting in unnecessary energy consumption in the physiological environment, thus significantly affecting their luminescence and photothermal conversion efficiency. However, photosensitizers with aggregation-induced emission (AIE) properties, as an advanced conceptual visual processing platform, can effectively avoid molecular stacking by combining molecules with rotors and twisted structures. Controlled molecular rotation in the aggregated state can subtly modulate fluorescence intensity and photothermal efficiency. Therefore, developing a novel, highly efficient photosensitizer with AIE properties to reshape the tumor microenvironment using novel phototherapy-induced aging methods for tumor immunotherapy based on aging is a pressing issue for the treatment of TNBC. Summary of the Invention

[0005] To address the shortcomings and deficiencies of existing technologies, the primary objective of this invention is to provide an AIE photosensitizer that achieves near-infrared II emission by embedding a thiophene rotor into the receptor core to realize "receptor core-driven rotation." Another objective of this invention is to provide a multimodal phototherapy agent comprising this type of AIE photosensitizer and an aging-inducing molecule. A further objective of this invention is to provide applications of the aforementioned photosensitizer or multimodal phototherapy agent.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] This invention provides an AIE photosensitizer that emits light in the near-infrared II region by embedding a thiophene rotor into the acceptor core to achieve "acceptor core-driven rotation," and is selected from TDQ, fBDQ, and BDQ with the following structural formulas:

[0008]

[0009]

[0010]

[0011] This invention also provides a method for preparing the above-mentioned AIE photosensitizer, the reaction formula of which is shown below, including the following steps:

[0012]

[0013] (1) The dibromo core compound 1a or 1b, tributyl(2,3-dihydrothiopheno[3,4-b][1,4]dioxin-5-yl)stanane, and catalyst are refluxed in an organic solvent to give intermediate product 2a or 2b.

[0014] In this step, the catalyst includes Pd(PPh3)4, Ph2P(CH2)2PPh2(dppe), Ph2P(CH2)3PPh2(dppp), etc., preferably Pd(PPh3)4; the organic solvent includes one or more mixed solvents such as toluene, xylene, and chloroform, preferably toluene; the molar ratio of dibromo core compound 1a or 1b, tributyl(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)stanane, and the catalyst is preferably 2.4-3:6-8:0.15-0.2; the reflux reaction time is preferably 42-54 hours.

[0015] Furthermore, after the reaction is completed, the step also includes a purification step: after the reaction solution is cooled, the solvent is removed by rotary evaporation, and then purified by silica gel column chromatography (eluent: n-hexane) to obtain the target product.

[0016] (2) Intermediate product 2a or 2b and brominated reagent are reacted in an organic solvent to obtain intermediate product 3a or 3b.

[0017] In this step, the brominating reagent includes N-bromosuccinimide (NBS), tetrabutylammonium tribromide, pyridinium bromide, etc., preferably NBS; the organic solvent includes one or more mixed solvents selected from toluene, xylene, chloroform, acetic acid, formic acid, etc., preferably a mixed solvent composed of chloroform and acetic acid in a volume ratio of 1:1; the molar ratio of intermediate product 2a or 2b to NBS is preferably 0.3-0.4:6-8; the reaction temperature is preferably room temperature, the reaction time is preferably 8-16 hours, and the reaction is preferably carried out under stirring.

[0018] Further, the step is as follows: under a nitrogen (N2) atmosphere, intermediate product 2a or 2b is dissolved in an organic solvent to obtain solution 1; under light-protected conditions, N-bromosuccinimide (NBS) is dissolved in an organic solvent to obtain solution 2; solution 2 is added dropwise to solution 1 over 25–35 minutes to carry out the reaction. The preferred volume ratio of solution 1 to solution 2 is 18–22:8–12.

[0019] Furthermore, after the reaction is completed, a purification step is also included: the reaction solution is dried and purified by silica gel column chromatography to obtain the target product.

[0020] (3) Intermediate product 3a or 3b, N,N-diphenyl-4-(tributyltinyl)aniline or 9,9-dimethyl-10-(4-(tributyltinyl)phenyl)-9,10-dihydroacridine, and a catalyst are reacted in an organic solvent to obtain the AIE photosensitizer. Specifically, intermediate product 3a, N,N-diphenyl-4-(tributyltinyl)aniline, and a catalyst are reacted to obtain AIE photosensitizer BDQ; intermediate product 3a, 9,9-dimethyl-10-(4-(tributyltinyl)phenyl)-9,10-dihydroacridine, and a catalyst are reacted to obtain AIE photosensitizer fBDQ; intermediate product 3a, N,N-diphenyl-4-(tributyltinyl)aniline, and a catalyst are reacted to obtain AIE photosensitizer TDQ.

[0021] In this step, the catalyst includes Pd(PPh3)4, Ph2P(CH2)2PPh2(dppe), Ph2P(CH2)3PPh2(dppp), etc., preferably Pd(PPh3)4; the organic solvent includes one or more mixed solvents selected from toluene, xylene, chloroform, etc., preferably toluene; the molar ratio of intermediate product 3a or 3b, N,N-diphenyl-4-(tributyltinyl)aniline or 9,9-dimethyl-10-(4-(tributyltinyl)phenyl)-9,10-dihydroacridine and catalyst is preferably 1:2 to 2.5:0.03 to 0.1; the reaction temperature is preferably 90 to 105°C, the reaction time is preferably 42 to 54 h, and the reaction is preferably carried out under stirring.

[0022] Furthermore, after the reaction is completed, a purification step is also included: the reaction solution is cooled and poured into water, extracted with dichloromethane (DCM), the organic phase is washed successively with saturated potassium fluoride (KF) solution and saturated brine, dried with magnesium sulfate, the solvent is evaporated, and the residue is purified by silica gel column chromatography (eluent: dichloromethane / n-hexane, volume ratio 1:5) to obtain the target product.

[0023] The present invention also provides a composition comprising the above-mentioned AIE photosensitizer and an aging-inducing molecule. The aging-inducing molecule includes Aurora kinase inhibitors, CDK4 / 6 inhibitors, PARP inhibitors, etc. The Aurora kinase inhibitor includes alicritinib (Ali), VX-689, barasertib, etc. Preferably, the aging-inducing molecule is alicritinib (Ali).

[0024] Furthermore, the composition containing the above-mentioned AIE photosensitizer and aging-inducing molecule is nanoparticles co-loaded with the AIE photosensitizer and aging-inducing molecule.

[0025] Furthermore, the composition containing the above-mentioned AIE photosensitizer and aging-inducing molecule is a liposome co-loaded with the AIE photosensitizer and aging-inducing molecule.

[0026] The preparation of liposomes co-loaded with AIE photosensitizer and aging-inducing molecule includes the following steps: adding aging-inducing molecule, AIE photosensitizer, phospholipid PEG or its derivative to an organic solvent and sonicating, adding the resulting mixture to water and sonicating again, evaporating the organic solvent and dialyzing with water, and then isothermal drying to obtain liposomes co-loaded with AIE photosensitizer and aging-inducing molecule. The phospholipid PEG or its derivative includes DSPE-PEG2000, DSPE-TK-PEG2000, DSPE-PEG-MAL, DSPE-PEG3400, etc.; the organic solvent includes one or more mixed solvents selected from toluene, xylene, chloroform, dimethyl sulfoxide (DMSO), etc., preferably a mixed solvent composed of DMSO and chloroform in a volume ratio of 0.1-0.2:0.8-1.2; the preferred mass ratio of aging-inducing molecule, AIE photosensitizer, phospholipid PEG or its derivative is 0.8-1.2:0.8-1.2:3.5-4.5.

[0027] Further, the preparation of the liposomes co-loaded with AIE photosensitizer and aging-inducing molecule includes the following steps: a mixture containing aging-inducing molecule, AIE photosensitizer, phospholipid PEG or its derivative in a mass ratio of 0.8–1.2:0.8–1.2:3.5–4.5, and DMSO:chloroform in a volume ratio of 0.1–0.2:0.8–1.2 is sonicated at an output power of 10–14 W. Subsequently, under sonication conditions, the resulting mixture is rapidly injected into water and sonicated continuously for 1.5–2.5 minutes. After evaporating chloroform by ventilation, the suspension is dialyzed with water using a membrane with a molecular weight cutoff of 100 kDa for 18–32 hours, and then freeze-dried to obtain the liposomes co-loaded with AIE photosensitizer and aging-inducing molecule.

[0028] The present invention also provides the application of the above-mentioned AIE photosensitizer or the above-mentioned composition containing AIE photosensitizer and aging-inducing molecule in the preparation of multimodal phototherapy agents.

[0029] A multimodal phototherapy agent comprising the above-mentioned AIE photosensitizer or the above-mentioned composition containing the AIE photosensitizer and the aging-inducing molecule.

[0030] This invention also provides the use of the above-mentioned AIE photosensitizer, or the above-mentioned composition containing the AIE photosensitizer and aging-inducing molecule, or the above-mentioned multimodal phototherapeutic agent in the preparation of tumor diagnostic reagents or tumor therapeutic drugs. The tumor therapeutic drugs include at least one of the following: tumor prevention / treatment drugs, photothermal / photodynamic therapy drugs for tumors, immunotherapy drugs for tumors, and drugs for treating primary tumors, tumor recurrence, and re-challenge.

[0031] A tumor diagnostic reagent or tumor therapeutic drug comprising the above-mentioned AIE photosensitizer or the above-mentioned composition containing AIE photosensitizer and aging-inducing molecule or the above-mentioned multimodal phototherapy agent.

[0032] The present invention has the following advantages and effects compared with the prior art:

[0033] (1) The self-assembled aggregation-induced emission phototherapy agent TDQ and the aging-inducing molecule Ali multimodal phototherapy agent of the present invention have excellent properties such as uniform size distribution, good stability, strong ability to target cancer cells, good biocompatibility, and effective ROS response to release drugs in cancer cells.

[0034] (2) The near-infrared II luminescent AIE phototherapy agent constructed in this invention has near-infrared II fluorescence signal, photodynamic properties, high photothermal conversion efficiency and good photostability under 808 nm excitation.

[0035] (3) The self-assembled phototherapy agent TDQ and the aging-inducing molecule Ali designed in this invention are multimodal phototherapy agents that can achieve efficient drug delivery to tumor tissue and ROS-responsive release. Their PDT / PTT induces immunogenic death of 4T1 cells, and Ali drugs effectively drive cellular senescence by inhibiting mitosis and regulating the senescence-associated secretory phenotype (SASP), thereby enhancing the sensitivity of cancer cells to reactive oxygen species damage. Under this dual action, they can effectively and powerfully recruit and activate antigen-presenting cells, enhancing anti-tumor immunity. Ultimately, this achieves the inhibition of distant tumors and tumor recurrence.

[0036] (4) The self-assembled phototherapy agent TDQ and the aging-inducing molecule Ali multimodal phototherapy agent of the present invention also have the advantages of low toxicity and good biocompatibility.

[0037] (5) The preparation process of the self-assembled phototherapy agent TDQ and the aging-inducing molecule Ali of the present invention is simple and low-cost, and is suitable for large-scale production and clinical medical use. Attached Figure Description

[0038] Figure 1The images show the photophysical properties of TDQ, fBDQ, and BDQ. Among them, (a) is the absorption spectrum of TDQ, fBDQ, and BDQ in aqueous solution; (b) is the fluorescence (FL) spectrum of TDQ, fBDQ, and BDQ in aqueous solution; and (c) is the fluorescence intensity ratio (F / F0) of TDQ, fBDQ, and BDQ at different DMSO / water volume ratios.

[0039] Figure 2 This study investigates the photothermal properties of TDQ, fBDQ, and BDQ. (a) shows the photothermal properties of TDQ, fBDQ, and BDQ at 1 W / cm². 2 (a) Temperature variation of TDQ under 808 nm laser irradiation; (b) Temperature variation of TDQ at different concentrations under laser irradiation (808 nm, 1 W / cm²). 2 ( ) represents the photothermal effect under the conditions of heating and cooling; (ce) is a graph showing the temperature changes of BDQ, fBDQ, and TDQ during the three heating and cooling processes.

[0040] Figure 3 This is the electron spin resonance (ESR) signal spectrum of TDQ. Where (ac) represent singlet oxygen (…). 1 O2), superoxide anion (·O2) - ) and hydroxyl radicals (·OH). TDQ concentration was 100 μM, laser conditions were 808 nm, 1 W / cm 2 .

[0041] Figure 4 The structure of Ali / TDQ-NP is characterized. Among them, (a) is the UV absorption spectrum of Ali / TDQ-NP, TDQ-NP, and Ali-NP; (b) is the hydrated particle size of Ali / TDQ-NP and (c) is the transmission electron microscope (TEM) image of Ali / TDQ-NP, scale bar: 100 nm; (d) is the zeta potential of Ali / TDQ-NP and unloaded blank nanoparticles; (e) is the hydrated particle size of Ali / TDQ-NP after incubation in different media for different times.

[0042] Figure 5 This is a graph showing the survival rate of 4T1 cells after treatment with different agents.

[0043] Figure 6 This is a detection of ATP and HMGB1 release concentrations and calreticulin (CRT) expression results in 4T1 cells after treatment with different agents. Among them, (a) is a confocal microscopy image of calreticulin (CRT) in 4T1 cells, and (b, c) are graphs showing the content expression of HMGB-1 and ATP after different treatments.

[0044] Figure 7The results show the senescence and cell cycle arrest of tumor cells induced by phototherapy. (a, b) are representative images of 4T1 cells after treatment with different agents and a statistical diagram of SaβG positive cells; (c) are the Western blot results of p21 and p16 protein expression in 4T1 cells after treatment with different agents; (d) are the flow cytometry analysis diagrams of cell cycle distribution after treatment with different nanoparticles (NPs).

[0045] Figure 8 This is a graph showing the results of senescence-induced SASP promoting APC activation and enhancing tumor immunotherapy. Among them, (a) is a flow cytometry image of mature dendritic cells (DCs) in different treatment groups; (b) is a quantitative analysis of mature dendritic cells after different treatments; (ce) shows the expression results of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) released by DCs in different treatment groups.

[0046] Figure 9 These are tumor growth curves and tumor weight curves of mice treated with different formulations in the 4T1 tumor-bearing mouse model, as well as tumor weight curves collected and measured on day 21.

[0047] Figure 10 These are tumor growth curves of mice treated with different preparations in a postoperative recurrence model, and tumor weight curves of mice collected and measured on day 21.

[0048] Figure 11 This is a graph showing the weight growth of mice after treatment with different formulations.

[0049] Figure 12 This is a schematic diagram illustrating the light-controlled responsive drug release achieved by the present invention through the co-loading of TDQ with the aging-inducing drug Ali using the ROS-responsive carrier DSPE-TK-PEG. Detailed Implementation

[0050] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the reagents, biological materials, etc. used in the following examples are commercially available.

[0052] The 1,2-distearate-sn-glycerol-3-phosphorylethanolamine-thioketal-polyethylene glycol 2000 (DSPE-TK-PEG2000) used in the following examples was purchased from Shanghai Yarui Biotechnology Co., Ltd. (China). DAPI was purchased from Sigma-Aldrich (USA). Alicetinib (Ali), ELISA kits, and 1640 cell culture medium were purchased from Guangzhou Ruiao Biotechnology Co., Ltd. (China). All aqueous solutions were prepared using deionized (DI) water purified by a purification system (Direct-Q3, Millipore, USA). Other solvents used were purchased from Sinopharm Chemical Reagent Co., Ltd. (China) and Shanghai Maclean Biotechnology Co., Ltd. (China).

[0053] The tumor cells used in the following examples were obtained from the 4T1 mouse breast cancer cell line from the Cell Bank of the Chinese Academy of Sciences and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum at a humidified atmosphere of 37°C.

[0054] Female BALB / c animals aged 5-6 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0055] Example 1

[0056] (I) Preparation of photosensitizers that emit light in the AIE near-infrared II region

[0057] (1) Synthesis of intermediate product 3a or 3b

[0058] Add 0.28 mmol of dibromo-based core compound 1a (4,9-dibromo-6,7-diphenyl-benzo[C][1,2,5]thiadiazo[3,4-G]quinoxaline, CAS: 1262727-09-2) or 1b (4,9-dibromo-6,7-bis(thiophen-2-yl)-[1,2,5]thiadiazo[3,4-G]quinoxaline, CAS: 2196162-74-8), 0.7 mmol of tributyl(2,3-dihydrothiopheno[3,4-b][1,4]dioxin-5-yl)stanane, 0.017 mmol of Pd(PPh3)4, and 20 mL of toluene to the flask. After refluxing the mixture at 120°C for 48 hours, the system was cooled to room temperature, the solvent was removed by rotary evaporation, and then purified by silica gel column chromatography (eluent: n-hexane) to obtain compound 2a or 2b (yield: 55-65%).

[0059] Under a nitrogen atmosphere, 0.33 mmol of compound 2a or 2b was dissolved in 20 mL of a 1:1 mixture of CHCl3 and acetic acid. At room temperature and in the dark, 6.6 mmol of N-bromosuccinimide (NBS) was dissolved in 10 mL of a 1:1 mixture of chloroform and acetic acid and slowly added dropwise to the above system over 30 minutes. After stirring the mixture overnight, it was dried by compressed air and then purified by silica gel column chromatography with n-hexane to give compound 3a or 3b (yield: 80–90%).

[0060] (2) Synthesis of BDQ, fBDQ and TDQ

[0061] Subsequently, 0.07 mol of compound 3a, 0.15 mmol of N,N-diphenyl-4-(tributyltinyl)aniline (CAS: 454182-37-7), and 3 μmol of Pd(PPh3)4 were dissolved in 10 mL of toluene, and the mixture was stirred at 100 °C for 48 hours. After cooling the system to room temperature, the mixture was poured into water and extracted with dichloromethane (DCM). The organic phase was washed successively with saturated KF solution and saturated brine, and then dried over MgSO4. After solvent evaporation, the residue was purified by silica gel column chromatography (eluent: dichloromethane / n-hexane, v / v ratio 1:5) to obtain BDQ.

[0062] Using compound 3a and 9,9-dimethyl-10-(4-(tributyltinyl)phenyl)-9,10-dihydroacridine as reactants, fBDQ was obtained via a route similar to that used in the synthesis of BDQ.

[0063] Using compound 3b and N,N-diphenyl-4-(tributyltinyl)-aniline as reactants, TDQ was obtained via a route similar to that used in the synthesis of BDQ.

[0064] The yield of the above products is 25-35%.

[0065] II. Characterization of AIE molecules

[0066] The optical properties of the prepared BDQ, fBDQ, and TDQ were tested. The absorption spectrum of the AIE molecule was measured using a UV-spectrum spectrophotometer, and the fluorescence spectrum was measured using a fluorescence spectrometer. Figure 1 The UV-Vis absorption spectroscopy revealed that BDQ, fBDQ, and TDQ exhibited maximum absorption peaks at 692 nm, 633 nm, and 733 nm in their aggregated aqueous solutions. The maximum fluorescence emission values ​​for BDQ, fBDQ, and TDQ occurred at 993 nm, 1015 nm, and 1025 nm, respectively, highlighting their significant potential for deep tissue imaging in the near-infrared II region.

[0067] III. Photothermal and Photodynamic Properties of AIE Molecules

[0068] (1) An 808 nm near-infrared laser purchased from Changchun Xingong Technology Co., Ltd. was used, with a wavelength of 1 W / cm². 2 Water containing 0, 25, 50, and 100 μM TDQ was irradiated with different power levels. The temperature of the TDQ during laser irradiation was recorded every 30 seconds using an infrared thermal imaging system.

[0069] (2) Measure the irradiance power (1 W / cm) using an infrared thermal imaging system. 2 Heating curves of different AIE molecules for three cycles under 808 nm laser irradiation.

[0070] (3) Detection of singlet oxygen using electron spin resonance (ESR) spectroscopy. 1 O2), superoxide anion (·O2) - The generation of hydroxyl radicals (·OH) and hydroxyl radicals (·OH).

[0071] The results are as follows Figure 2 As shown in figure a, among the three AIE molecules, TDQ exhibits the strongest photothermal effect at a concentration of 100 μM. Meanwhile, TDQ at 1 W / cm²... 2 The temperature rapidly rises to 55°C within 5 minutes after irradiation with an 808 nm laser; its effect is concentration-dependent. Figure 2 b). Furthermore, by maintaining a consistent temperature throughout three on-off heating cycles, the photothermal stability and resistance to photobleaching of the three AIE molecules (100 μM) were confirmed. Figure 2 ce).

[0072] The generation mechanism of reactive oxygen species (ROS) was investigated using electron spin resonance (ESR) spectroscopy. The procedure was as follows: The sample was dissolved in water and placed in a quartz sample tube. Magnetic field calibration was performed using standard samples (such as DPPH, diphenylpicrylhydrazine radical). The scan range was set to 3300-3450 G, power 1 mW, modulation amplitude 0.1-1 G, modulation frequency 100 kHz, and scan time 30 seconds. The prepared sample tube was vertically placed in the center of the instrument's sample chamber, ensuring the sample was within a uniform magnetic field region. Results are as follows: Figure 3 As shown, under illumination conditions of 808 nm wavelength and 1 W / cm² power density, 100 μM concentration of TDQ simultaneously produces singlet oxygen (…). 1 O2), superoxide anion (·O2) - ) and hydroxyl radicals (·OH).

[0073] Example 2

[0074] I. Preparation of self-assembled nanomaterials of AIE photosensitizers and aging molecules

[0075] Preparation of alixetinib (Ali) co-loaded liposomes with AIE (Ali / TDQ-NP), TDQ-loaded liposomes (TDQ-NP), and Ali-loaded liposomes (Ali-NP): A mixture containing 1 mg Ali, 1 mg TDQ, 4 mg DSPE-TK-PEG2000, 0.1 mL dimethyl sulfoxide (DMSO), and 1 mL chloroform was sonicated at 12 W. Subsequently, the resulting mixture was rapidly injected into 9 mL of water under 12 W sonication for 2 minutes. After stirring in a fume hood for 30 minutes to evaporate the chloroform, the suspension was dialyzed against water for 24 hours using a membrane with a molecular weight cutoff of 100 kDa. The final product, Ali / TDQ-NP, was obtained by freeze-drying. Ali or TDQ were encapsulated using the same method to obtain Ali-NP and TDQ-NP, respectively.

[0076] II. Characterization of Nanomaterials

[0077] The absorption spectra of AIE nanoparticles were measured using a UV-Vis spectrophotometer. The morphology of the nanoparticles was examined using a transmission electron microscope. The particle size and zeta potential of the samples were determined by DLS.

[0078] like Figure 4 Transmission electron microscopy (TEM) images show that the Ali / TDQ-NP nanoparticles (NPs) are uniformly spherical. The Ali / TDQ-NP nanoparticles are relatively dispersed and have a relatively uniform size distribution. Dynamic light scattering (DLS) measurements indicate that the Ali / TDQ-NP particle size is approximately 105 nm. The zeta potential of Ali / TDQ-NP is approximately -22.5 ± 0.8 mV, which is similar to the zeta potential (-22.8 ± 1.9 mV) of blank nanoparticles containing only DSPE-TK-PEG. The UV-Vis absorption spectrum of Ali / TDQ-NP nanoparticles shows absorption peaks at approximately 290 nm and 730 nm, indicating that the two compounds have been successfully co-loaded.

[0079] Example 3: Effects of Ali / TDQ-NP on 4T1 tumor cells

[0080] 4T1 cells were seeded in six-well plates (5000 cells per well) and incubated for 24 hours; then the original medium was replaced with fresh medium containing nanoparticles, and the cells were treated as follows: (1) PBS, (2) NIR (808 nm, 1 W / cm²). 2(3) TDQ-NP, (4) Ali-NP, (5) TDQ-NP + NIR, (6) Ali / TDQ-NP + NIR. Two hours after nanoparticle treatment, the NIR-treated groups were irradiated with an 808 nm laser (1 W / cm²). 2 After incubation for 48 hours, the viability of 4T1 cells was measured using the MTT assay (Beyotime Biotech. Inc.).

[0081] MTT cell viability assay results ( Figure 5 The results showed that TDQ-NP irradiated with laser produced significant cytotoxicity, with a cell survival rate of 33.86%; while the Ali / TDQ-NP+NIR group induced more than 90% of cancer cells to die.

[0082] Example 4: Ali / TDQ-NP induces immunogenic death in 4T1 tumor cells

[0083] 4T1 cells in 48-well plates (2 × 10⁴ cells per well) 4 After seeding cells for 12 hours, the following treatments were performed: (1) PBS, (2) NIR (808 nm, 1 W / cm) 2 (3) TDQ-NP, (4) Ali-NP, (5) TDQ-NP + NIR, (6) Ali / TDQ-NP + NIR. After treating cells with a culture medium containing nanoparticles for 2 hours, the NIR-treated groups were then irradiated with an 808 nm laser (1 W / cm²). 2 Cells were washed three times with PBS, fixed with 4% PFA, and permeabilized with 0.1% Triton X-100 for 10 minutes. After washing three times with PBS, cells were blocked with 10% FBS and incubated with anti-calreticulin (CRT) / FITC conjugate antibody (Bioss) for 30 minutes. Cells were washed three times with PBS and stained with DAPI for 20 minutes. Finally, cells were washed three times with PBS and observed using CLSM. Strong fluorescence signals of CRT were detected in the TDQ-NP and Ali / TDQ-NP treatment groups under near-infrared laser irradiation. Figure 6 a).

[0084] 4T1 cells were seeded into 12-well plates (2 × 10⁻⁶ cells per well). 5 Cells were cultured in wells (1 cell / well) and then treated as follows on the second day: (1) PBS, (2) NIR (808 nm, 1 W / cm) 2(3) TDQ-NP, (4) Ali-NP, (5) TDQ-NP + NIR, (6) Ali / TDQ-NP + NIR. After treating cells with a culture medium containing nanoparticles for 2 hours, the NIR-treated groups were then irradiated with an 808 nm laser (1 W / cm²). 2 , 5 min). To quantitatively detect HMGB1 release in the culture medium, the culture medium was collected 24 h after cell treatment. Then, according to the kit instructions, the levels of HMGB1 and ATP were detected by ELISA and ATP assay kits. Compared with the control group, the Ali / TDQ-NP+NIR group showed increased HMGB1 release and increased ATP levels in tumor cells. Figure 6 (b and 6c) indicate that Ali / TDQ-NP induced immunogenic death in tumor cells.

[0085] Example 5: Ali / TDQ-NP induces tumor cell senescence

[0086] (1) Cell senescence characteristic map: 4T1 cells were seeded into 12-well plates (2×10⁻⁶ cells per well). 5 Cells were cultured in wells (1 cell / well) and then treated as follows on the second day: (1) PBS, (2) NIR (808 nm, 1 W / cm) 2 (3) TDQ-NP, (4) Ali-NP, (5) TDQ-NP + NIR, (6) Ali / TDQ-NP + NIR. Cells were treated with a nanoparticle-containing culture medium for 2 hours, and the NIR-treated group was then irradiated with an 808 nm laser (1 W / cm²). 2 (5 min). After 24 h of treatment, the samples were stained with SaβG and observed under a microscope.

[0087] (2) Activation of the senescence pathway in 4T1 cells was verified by Western blot: 4T1 cells were seeded into 12-well plates (2×10⁻⁶ cells / wells). 5 Cells were cultured in wells (1 cell / well) and then treated as follows on the second day: (1) PBS, (2) NIR (808 nm, 1 W / cm) 2 (3) TDQ-NP, (4) Ali-NP, (5) TDQ-NP + NIR, (6) Ali / TDQ-NP + NIR. After treating cells with a culture medium containing nanoparticles for 2 hours, the NIR-treated group was irradiated with an 808 nm laser (1 W / cm²). 2(5 min). After 24 h of treatment, cells from each group were collected, and total cellular protein was extracted using protein lysis buffer. Protein content was detected using BCA reagent. After electrophoresis, the separated proteins were transferred to a PVDF membrane. After PVDF membrane transfer, the membrane was blocked with milk for 2 hours. After blocking, diluted p21 primary antibody (Bioss) and anti-p16 antibody (Bioss) solutions (diluted according to the recommended concentration of each antibody) were added and incubated overnight. After washing, secondary antibody was added, and the membrane was exposed, developed, and photographed.

[0088] (3) Cell cycle detection of cell arrest: 4T1 cells were seeded into 12-well plates (2×10⁻⁶ cells / wells). 5 Cells were cultured in wells (1 cell / well) and then treated as follows on the second day: (1) PBS, (2) NIR (808 nm, 1 W / cm) 2 (3) TDQ-NP, (4) Ali-NP, (5) TDQ-NP + NIR, (6) Ali / TDQ-NP + NIR. Cells were treated with a nanoparticle-containing culture medium for 2 hours, and the NIR-treated group was then irradiated with an 808 nm laser (1 W / cm²). 2 (5 min). After 24 h of treatment, cells from each group were collected and analyzed using a cell cycle kit.

[0089] Senescence-associated β-galactosidase (SAβG) activity was observed in 4T1 cells treated with different formulations, and TDQ-NP+NIR treatment also induced a considerably high level of SAβG. Figure 7 (a and 7b). Cell cycle analysis showed that, compared with the control group, the Ali-NP treatment group caused significant G2 / M phase arrest, consistent with its known mechanism of action as an inhibitor of aurora kinase A. The Ali / TDQ-NP + near-infrared (NIR) group showed even more pronounced G2 phase arrest, accompanied by mitotic catastrophe (…). Figure 7 d). Cell cycle-dependent kinase inhibitors p21CDKN1A and p16INK4a were identified as key molecules mediating tumor suppressor senescence (TIS) effects through different pathways. p21 arrests cell cycle progression upon p53 activation, while p16INK4a establishes stable cell cycle arrest. Western blot analysis showed that the expression of p21 and p16 was significantly upregulated after treatment with Ali-NP and DQ-NP+NIR. Figure 7 c).

[0090] Example 6: Ali / TDQ-NP induces DC cell maturation and inflammatory cytokine release

[0091] (1) Flow cytometry verification of the effect of Ali / TDQ-NP on DC cell maturation: BMDCs were isolated from the bone marrow of 8-week-old BALB / c mice. The cells were grouped and treated with 1×10⁻⁶ cells according to the above method. 5 12 h of 4T1 cells, then cultured in a Transwell system with 1×10 6 BMDCs were co-cultured for 12 h, stained with anti-CD80 and anti-CD86 (Biolegend), and then classified using flow cytometry (Beckman-Coulter, USA). Cytokines secreted by BMDCs were detected using an ELISA kit (Elabscience Biotechnology Co., Ltd.).

[0092] (2) Detection of inflammatory factors: 4T1 cells were seeded into 12-well plates (2×10⁻⁶ cells / wells). 5 Cells were cultured in wells (1 cell / well) and then treated as follows on the second day: (1) PBS, (2) NIR (808 nm, 1 W / cm) 2 (3) TDQ-NP, (4) Ali-NP, (5) TDQ-NP + NIR, (6) Ali / TDQ-NP + NIR. After treating cells with a culture medium containing nanoparticles for 2 hours, the NIR-treated groups were then irradiated with an 808 nm laser (1 W / cm²). 2 (5 min). To quantitatively detect inflammatory factors released in the culture medium, the culture medium was collected 24 h after cell treatment. Then, according to the kit instructions (Elabscience Biotechnology Co., Ltd.), 20 μL of culture medium was taken and the levels of IL-6, TNF-α, and IFN-γ were detected by ELISA.

[0093] 4T1 cells pretreated with Ali / TDQ-NP+ near-infrared light (NIR) showed a significant increase in the expression levels of maturation markers CD86 and CD80 on the surface of dendritic cells (DCs). Figure 8 (a, 8b). These activated dendritic cells (DCs) also secrete higher levels of pro-inflammatory cytokines, including IL-6, TNF-α, and IFN-γ. This result is consistent with the activated state and pro-inflammatory phenotype of dendritic cells (DCs), which may contribute to their antigen-presenting function and T-cell activation. Figure 8 ce).

[0094] Example 7: Tumor-inhibiting effect of Ali / TDQ-NP on 4T1 tumor-bearing mice after treatment

[0095] BALB / c mice were subcutaneously injected with 3×10⁻⁶ spores into the right back. 64 T1 cells were introduced to the primary tumor. When the tumor grew to approximately 200 mm... 3 Day 0 was designated as day 0 for treatment. Mice were randomly divided into 6 different groups (each group consisting of 5 mice): (1) PBS, (2) NIR (808 nm, 1 W / cm²). 2 (3) TDQ-NP, (4) Ali-NP, (5) TDQ-NP + NIR, (6) Ali / TDQ-NP + NIR. Twelve hours after tail vein injection of nanoparticles, mice treated with NIR were irradiated with laser (PTT / PDT). Body weight and tumor volume were monitored in all groups every 3 days. Tumor length and width were measured with calipers, and tumor volume was calculated using the following formula: Tumor volume = Tumor length × Tumor width. 2 / 2.

[0096] Tumor monitoring results showed that although both monotherapy groups (TDQ-NP+NIR and Ali-NP) exhibited some anti-tumor effects, the Ali / TDQ-NP+NIR group inhibited tumor growth, with tumor volume reduction of approximately 15-fold compared to the PBS control group, demonstrating effective inhibition of the primary tumor. Figure 9 ).

[0097] Example 8: Effects of Ali / TDQ-NP on tumor recurrence and metastasis in 4T1 tumor-bearing mice after treatment

[0098] BALB / c mice were subcutaneously injected with 5×10⁻⁶ mol / L on the right back. 6 4T1 cells, tumor volume reached 200 mm. 3 Treatment was then initiated. Mice were randomly divided into 6 different groups (each group consisting of 5 mice): (1) PBS, (2) NIR (808 nm, 1 W / cm²). 2 (3) TDQ-NP, (4) Ali-NP, (5) TDQ-NP + NIR, (6) Ali / TDQ-NP + NIR. PTT / PDT were performed on the NIR-treated groups 12 hours after nanoparticle treatment. The primary tumor on the right side of the mouse was then removed 4 days after treatment, and 5×10⁻⁶ NIR was injected again into the left hind leg of the mouse. 6 Four T1 cells were used to form secondary tumors. Tumor growth and mouse body weight were recorded at regular time intervals during recurrence and metastasis. Tumor length and width were measured with calipers, and tumor volume was calculated using the formula (tumor volume = tumor length × tumor width). 2 / 2). The mice were euthanized 21 days after treatment.

[0099] Tumor volume monitoring results showed that, compared with other groups, Ali / TDQ-NP + NIR treatment effectively inhibited the malignant growth of recurrent and metastatic tumors. Figure 10 Additionally, such as Figure 11 As shown, there were no abnormal changes in the body weight of mice in each group after different treatments.

[0100] In summary, the reactive oxygen species (ROS) responsive nanoparticles (Ali / TDQ-NP) formed by the self-assembly of the TDQ molecule and the Aurora kinase inhibitor alicritinib (Ali) of this invention retain AIE properties in the near-infrared II region while directing excited-state energy to non-radiative decay. This optimization significantly improves photothermal conversion efficiency and enhances the generation of type I and type II ROS under 808 nm irradiation, thereby enabling simultaneous tumor imaging and ablation. Therefore, Ali / TDQ-NP can mediate phototherapy response and induce mitochondrial dysfunction and oxidative stress, while promoting the targeted release of Ali. This dual mechanism effectively drives cellular senescence by inhibiting mitosis and regulating SASP, thereby enhancing the sensitivity of cancer cells to ROS damage. Unlike single therapies that only induce apoptosis or partial cell cycle arrest, vaccination with senescent cells induced by Ali / TDQ-NP can strongly recruit and activate antigen-presenting cells, enhancing anti-tumor immunity. Furthermore, Ali / TDQ-NP-induced ICD effectively triggers the release of damage-associated molecular patterns (DAMPs), promotes adenosine triphosphate (ATP) release, calreticulin (CRT) translocation to the cell membrane surface, and expression of high-mobility group box 1 (HMGB1), synergistically enhancing aging-induced immune responses and promoting the generation of downstream immune stimuli and the secretion of inflammatory factors. Mouse models demonstrated complete eradication of the primary tumor and prevention of recurrence.

[0101] The above embodiments are only used to help illustrate the present invention. The implementation of the present invention is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered as equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. An AIE photosensitizer, characterized in that, Selected from TDQ, fBDQ, and BDQ, whose structural formulas are shown below: ； ； 。 2. The method for preparing the AIE photosensitizer according to claim 1, characterized in that, Includes the following steps: (1) The dibromo core compound 1a or 1b, tributyl(2,3-dihydrothiopheno[3,4-b][1,4]dioxin-5-yl)stanane, and catalyst are refluxed in an organic solvent to give intermediate product 2a or 2b; (2) Intermediate product 2a or 2b and brominated reagent are reacted in an organic solvent to give intermediate product 3a or 3b; (3) The intermediate product 3a or 3b, N,N-diphenyl-4-(tributyltinyl)-aniline or 9,9-dimethyl-10-(4-(tributyltinyl)phenyl)-9,10-dihydroacridine, and a catalyst are reacted in an organic solvent to obtain the AIE photosensitizer. The structural formulas of the dibromo core compound 1a or 1b, intermediate product 2a or 2b, and intermediate product 3a or 3b are shown below: 。 3. The method for preparing the AIE photosensitizer according to claim 2, characterized in that: The catalysts include Pd(PPh3)4, Ph2P(CH2)2PPh2(dppe), and Ph2P(CH2)3PPh2(dppp); The brominating agents include N-bromosuccinimide, tetrabutylammonium tribromide, and pyridinium bromide.

4. A composition comprising the AIE photosensitizer and aging-inducing molecule as described in claim 1, characterized in that: The aging-inducing molecules mentioned include Aurora kinase inhibitors, CDK4 / 6 inhibitors, and PARP inhibitors.

5. The composition according to claim 5, characterized in that: The aging-inducing molecule mentioned is alicatetinib.

6. The composition according to claim 5, characterized in that: The composition is nanoparticles containing the AIE photosensitizer and aging-inducing molecule as described in claim 1.

7. The composition according to claim 5, characterized in that: The composition is a liposome containing the AIE photosensitizer and aging-inducing molecule as described in claim 1.

8. The use of the AIE photosensitizer of claim 1 or the composition of any one of claims 4-7 in the preparation of multimodal phototherapy agents, tumor diagnostic reagents or tumor therapeutic drugs.

9. The application according to claim 8, characterized in that: The tumor treatment drugs mentioned include at least one of the following: tumor prevention / treatment drugs, photothermal / photodynamic therapy drugs for tumors, immunotherapy drugs for tumors, and drugs for treating primary tumors, tumor recurrence, and re-challenge.

10. A multimodal phototherapy agent, tumor diagnostic reagent, or tumor treatment drug, characterized in that: It comprises the AIE photosensitizer of claim 1 or the composition of any one of claims 4-7.

Images