Near-infrared AIE light diagnosis and treatment agent with D-A-D structure as well as preparation method and application of near-infrared AIE light diagnosis and treatment agent

Abstract

The invention discloses a near-infrared AIE light diagnosis and treatment agent with a D-A-D structure as well as a preparation method and application thereof. The near-infrared AIE light diagnosis and treatment agent comprises a compound with a structural formula as shown in a formula I, or pharmaceutically acceptable salt, solvate and tautomer of the compound, wherein R is one or more of O, S and Se; m is n, X is O, S or Se, n is equal to 0, 1, 2 or 3, and dotted lines represent joints. The TPATBS NPs provided by the invention has a relatively strong inhibition effect on the growth of tumor cells, and has a good anti-tumor effect. Experimental results show that the optical diagnosis and treatment agent-TPATBS NPs can be applied to long-time tumor monitoring and tumor phototherapy with photodynamic and photo-thermal therapy synergy, multi-mode imaging guided tumor phototherapy is achieved, and a new thought is provided for diagnosis and treatment integrated application of AIE diagnosis and treatment agents.

Description

Technical Field

[0001] This invention relates to the field of medical technology, and in particular to a near-infrared AIE phototherapy agent with a DAD structure, its preparation method, and its application. Background Technology

[0002] Cancer, a malignant tumor originating from epithelial tissue, is characterized by high spread, uncontrollability, and high mortality, seriously threatening human health. Efficient tumor diagnosis and treatment technologies are a significant challenge facing the medical field, making improving diagnostic efficiency and treatment outcomes a crucial scientific issue. Phototherapy, as a novel disease diagnosis and treatment technology, plays a vital role in tumor monitoring and in situ therapy. Bioimaging diagnostic methods mainly include fluorescence imaging (FLI), photoacoustic imaging (PAI), and photothermal imaging (PTI), offering advantages such as non-invasiveness, high sensitivity, rapid and efficient operation, low cost, and real-time monitoring. These methods have been widely applied in biomedical and clinical fields such as subcellular localization analysis and fluorescence surgical navigation. Phototherapy is a novel non-invasive tumor treatment method, primarily divided into photodynamic therapy (PDT) and photothermal therapy (PTT). PDT mainly utilizes phototherapeutic agents to generate reactive oxygen species (ROS) through energy conversion, thereby achieving a therapeutic effect. PTT utilizes a specific wavelength of light source to excite a photothermal agent, causing it to transition from its ground state to an excited state. The energy is converted into heat through vibrational relaxation, and the resulting heat raises the local temperature of biological tissues, thereby exerting a certain killing effect on tumor cells. It has high selectivity and spatiotemporal controllability, and activates certain immune functions in the body, thus achieving the effect of tumor treatment.

[0003] Aggregation-induced emission (AIE) materials are widely used in bioimaging and disease treatment due to their unique properties. As novel organic fluorescent materials, AIE materials are widely applied in bioimaging and tumor phototherapy due to their advantages such as easy aggregation and emission, high sensitivity, good photostability, large Stokes shift, low background noise, low toxicity, and strong biovisualization capabilities. In recent years, researchers have been continuously exploring and developing multifunctional novel phototherapy agents. Among them, by cleverly designing molecular pathways to balance excited-state energy dissipation, a multimodal imaging-guided, synergistic strategy for tumor diagnosis and treatment can be achieved. Energy dissipated by radiative decay can achieve free light emission (FLI), while energy dissipated through some non-radiative decay pathways is used for phototherapy (PAI), phototherapy (PTI), and phototherapy (PTT), and energy dissipated through intersystem crossing (ISC) pathways is used for phototherapy (PDT). The synergy of multiple tumor diagnosis and treatment methods improves the efficacy of tumor diagnosis and treatment. Therefore, the design and optimization of novel fluorescent molecules to construct new tumor diagnosis and treatment systems is of great significance for tumor diagnosis and treatment. Summary of the Invention

[0004] Purpose of the invention

[0005] To overcome the above shortcomings, the present invention aims to provide a near-infrared AIE phototherapy agent with a DAD structure, its preparation method, and its application. The near-infrared AIE phototherapy agent of the present invention has a D-π-A-π-D structure. This phototherapy agent exhibits excellent near-infrared fluorescence emission characteristics, a large Stokes shift, suitable drug delivery size, and stability. Under photoactivation, it can generate different types of reactive oxygen species and possesses high photothermal conversion efficiency and photothermal cycling stability. It also has a significant inhibitory effect on tumor cell growth, providing new ideas for the design and development of multifunctional AIE phototherapy agents and their integrated multimodal tumor diagnosis and treatment applications.

[0006] Solution

[0007] To achieve the objectives of this invention, the technical solution adopted is as follows:

[0008] In a first aspect, the present invention provides a near-infrared AIE phototherapy agent having a DAD structure, comprising a compound with the structural formula shown in Formula I, or a pharmaceutically acceptable salt thereof, a solvate thereof, and a tautomer thereof;

[0009]

[0010] Wherein, R is one or more of O, S, and Se;

[0011] M is n Where X represents O, S, or Se, n = 0, 1, 2, or 3, and the dashed line represents the connection point.

[0012] Furthermore, n is either 1 or 2.

[0013] Furthermore, it includes compounds with the following structural formulas, or pharmaceutically acceptable salts thereof, or solvates thereof, or tautomers thereof:

[0014]

[0015] Furthermore, it is a nanotherapeutic agent, optionally formed by coating a compound as shown in Formula I and an amphiphilic polymer coating agent via a nano-co-precipitation method;

[0016] Optionally, the external structure of the near-infrared AIE phototherapy agent is a DSPEG-mPEG2000 or Pluronic F127 amphiphilic polymer coating agent, and the core structure is a aggregation-induced emission material TPATBS that generates heat under laser excitation, forming an aggregation-induced emission phototherapy agent TPATBS NPs.

[0017] In a second aspect, a method for preparing the near-infrared AIE phototherapy agent described in the first aspect is provided, comprising the following steps: in a solvent, under the action of an alkaline substance and Pd(OAc)2, the compounds represented by formula A and formula B generate the compound represented by formula I:

[0018]

[0019] The M and R in this case are the same as those in compound I.

[0020] Optionally, the reaction formula for a specific compound TPATBS is as follows:

[0021]

[0022] Furthermore, the solvent is selected from one or more of toluene, acetonitrile, dimethyl sulfoxide, and dimethylformamide;

[0023] And / or, the alkaline substance is selected from one or more of potassium carbonate, sodium carbonate, and cesium carbonate;

[0024] And / or, the reaction is carried out in an inert gas atmosphere, optionally with nitrogen;

[0025] And / or, the reaction temperature is 40–120°C, optionally 80–120°C, optionally 100–110°C;

[0026] And / or, the reaction time is 10 to 18 hours, optionally 12 to 18 hours;

[0027] And / or, the molar ratio of the compounds shown in Formula A and Formula B is 1:0.9 to 1.5, optionally 1:1 to 1.5, optionally 1:1 to 1.2.

[0028] Furthermore, the compound shown in Formula I is prepared into nanoparticles: the compound shown in Formula I and the amphiphilic polymer are dissolved in an organic solvent, and nanoparticles are obtained by sonication, dialysis, and filtration.

[0029] Furthermore, the amphiphilic polymer is Pluronic F127 or DSPEG-mPEG-2000;

[0030] Optionally, the mass ratio of the compound shown in Formula I to the amphiphilic polymer is 1:30 to 1:150, optionally 1:30 to 1:100, optionally 1:30 to 1:50, optionally 1:40, optionally 1:100 to 150, optionally 1:100.

[0031] Furthermore, the organic solvent is selected from one or more of THF, ACN, DMSO, and DMF;

[0032] Optionally, the diameter of the nanoparticles is 100–150 nm;

[0033] Optionally, the ultrasound time is 1–50 min, or optionally 30–40 min;

[0034] Optionally, the ultrasonic power is 20-300W;

[0035] Optionally, dialysis is performed in deionized water, and the deionized water is changed every 6 to 10 hours. Optionally, dialysis is performed for 24 to 56 hours, or 48 to 56 hours.

[0036] Optionally, the pore size of the filter is 150–200 μM, optionally 200 μM.

[0037] The present invention provides a method for preparing a photothermal therapeutic agent with AIE properties as described above, comprising the following steps (taking the preferred synthesis of TPATBS molecule as an example): 4,7-bis(5-bromo-2-thienyl)-1,2,5-benzoselenide diazole, 4-(diphenylamino)phenylborone, Na2CO3, and Pd(OAc)2 are placed in a 250 mL round-bottom flask, and toluene is added. The temperature is adjusted to 100°C, and the mixture is stirred for 12 h under nitrogen protection, reacting overnight. After cooling to room temperature, water and chloroform are added. The organic layer is separated and washed, and dried with anhydrous MgSO4 to obtain the crude product. An eluent of petroleum ether / dichloromethane (v1 / v2 = 20 / 1) is prepared, and the solid TPATBS is purified by silica gel column chromatography. The successful synthesis of the molecule is detected by NMR and mass spectrometry, and its UV absorption peak and fluorescence emission peak are measured to be 560 nm and 688 nm, respectively.

[0038] Furthermore, the AIE phototherapy agent TPATBS exhibits a small amount of fluorescence signal in a tetrahydrofuran (THF) diluted solution. Upon addition of ultrapure water to the THF solution containing TPATBS, the fluorescence intensity gradually decreases, indicating that the TPATBS molecule possesses excellent fluorescence imaging capabilities and good AIE properties.

[0039] Thirdly, the invention provides an application of the near-infrared AIE phototherapy agent described in the first aspect or the near-infrared AIE phototherapy agent prepared by the preparation method described in the second aspect in the preparation of tumor diagnosis, treatment and integrated diagnosis and treatment or anti-tumor products. The products may be long-term (long-term can be 15 days or more) tumor monitoring probes, multimodal imaging contrast agents and superficial tumor phototherapy agents.

[0040] Furthermore, near-infrared AIE phototherapy agents are used under 635nm laser irradiation.

[0041] Optionally, the near-infrared AIE phototherapy agent can reach a photothermal temperature of 65-69°C under 635nm laser irradiation intensity, and optionally 67.5-69°C;

[0042] Optionally, the imaging capability of the near-infrared AIE phototherapy agent is maintained for 10 to 20 days, or optionally 12 to 15 days.

[0043] Fourthly, a diagnostic or treatment method is provided using the near-infrared AIE phototherapy agent described in the first aspect or the near-infrared AIE phototherapy agent prepared by the preparation method described in the second aspect, wherein an effective dose of the near-infrared AIE phototherapy agent is injected into a subject in need.

[0044] Beneficial effects

[0045] (1) The phototherapy agent TPATBS of the present invention has a DAD structure and is simple to synthesize, low in cost and easy to synthesize.

[0046] (2) The present invention can prepare core-shell structured nanoparticles by combining TPATBS with amphiphilic polymers. The core structure is aggregation-induced emission molecule TPATBS, which is encapsulated in amphiphilic polymers Pluronic F127 or DSPEG-mPEG2000 to form aggregation-induced emission polymers. The synthesis method is simple, low-cost and easy to synthesize.

[0047] (3) The phototherapy agent provided by the present invention has suitable drug delivery size, biocompatibility, stability and low biotoxicity, and can be safely applied to biological systems and accumulates in large quantities at the tumor site to achieve long-term tumor monitoring (up to 15 days).

[0048] (4) The phototherapy agent provided by this invention has a high ROS generation capacity and photothermal effect under laser irradiation, which can realize synergistic tumor phototherapy with photodynamic therapy and photothermal therapy. The phototherapy agent provided by this invention can be applied to the field of disease diagnosis and treatment, realizing long-term tumor monitoring and tumor phototherapy, and achieving the integrated effect of tumor diagnosis and treatment. Attached Figure Description

[0049] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0050] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of compound TPATBS from Example 1 of this invention.

[0051] Figure 2 This is the mass spectrum of compound TPATBS from Example 1 of the present invention.

[0052] Figure 3 The images show (a) UV absorption and (b) fluorescence emission spectra of compound TPATBS in THF solution from Example 1.

[0053] Figure 4 These are the AIE performance test results of compound TPATBS from Example 1 of the present invention, where (a) is the AIE performance curve of compound TPATBS, and (b) is the AIE performance curve of compound TPATBS with respect to the proportion of n-hexane in the mixed solution (f). w The change in fluorescence intensity (%).

[0054] Figure 5 This is a schematic diagram of the reaction process for preparing TPATBS NPs by the nano-coprecipitation method in Example 2 of the present invention.

[0055] Figure 6 These are the ultraviolet absorption spectrum and fluorescence emission spectrum of the TPATBS NPs in Example 2 of the present invention, wherein (a) is the ultraviolet absorption spectrum and (b) is the fluorescence emission spectrum.

[0056] Figure 7 This is an evaluation of the particle size and stability of TPATBS NPs in Example 2 of the present invention. (a) TEM image of TPATBS NPs; (b) hydrated particle size; (c) Zeta potential.

[0057] Figure 8The hydrated particle size changes of TPATBS NPs in Example 2 of this invention after being stored in different solvents (DMEM, FBS, PBS and ultrapure water) for different times (1 day, 7 days and 14 days), where (a) is 1 day, (b) is 7 days and (c) is 14 days.

[0058] Figure 9 This is the detection of the ROS generation capability of TPATBS NPs under illumination in Test Example 1 of the present invention.

[0059] Figure 10 This is the photothermal effect detection of Test Example 2 of the present invention, wherein (a) aqueous solutions of different concentrations of TPATBS NPs at 800 mW / cm 2 (a) Photothermal effect under laser irradiation; (b) Photothermal effect of 90 μM / L TPATBS NPs aqueous solution under laser irradiation with different optical power; (c) Physical images of TPATBS NPs aqueous solutions of different concentrations under laser irradiation with different optical power densities; (d) Photothermal cycle stability test; (e) Calculation of photothermal conversion efficiency.

[0060] Figure 11 This is the result of the test example 2 of the present invention, which evaluates the cytotoxicity of TPATBS NPs to MDA-MB-231 cells by different incubation times using the CCK-8 assay, where (a) 12h, (b) 24h, (c) 36h, and (d) 48h.

[0061] Figure 12 This describes the uptake of nanoparticles by TPATBS NPs and MDA-MB-231 cells after co-incubation for 2h, 4h, 6h, and 8h in Test Example 3 of this invention.

[0062] Figure 13 These are the imaging results of TPATBS NPs in Test Example 3 of the present invention at the animal level, wherein (a) is the imaging result of subcutaneous injection and (b) is the imaging result of tail vein injection.

[0063] Figure 14 These are the photothermal imaging results of TPATBS NPs in Test Example 4 of this invention at the animal level. (a) Temperature changes at the tumor site in mice; (b) Actual image.

[0064] Figure 15 The following are the changes in tumor volume (a), mouse weight (b), and HE staining results of the heart, liver, spleen, lung, and kidney of mice after treatment in each group of mice. Detailed Implementation

[0065] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0066] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, methods, means, etc., well-known to those skilled in the art, are not described in detail in order to highlight the spirit of the present invention.

[0067] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0068] Example 1: Synthesis and Characterization of Compound TPATBS

[0069] The general structural formula of this type of compound provided in this embodiment is:

[0070]

[0071] Where R represents O, S, and Se, and M represents n (n = 0, 1, 2, 3) elements. Let X be O, S, or Se.

[0072] These compounds are AIE molecules with a DAD structure, where M has one thiophene group. Taking R=S as an example, named TPATBSR, its preparation method includes the following steps:

[0073] 1. Synthesis of TPATBS compounds

[0074] (1) TPA (4-(diphenylamino)phenylboronic acid) (249.00 mg, 0.50 mmol), TBS (4,7-bis(5-bromo-2-thienyl)-2,1,3-benzoselenide diazole) (249.00 mg, 0.50 mmol), Na2CO3 (530.00 mg, 5.00 mmol) and Pd(OAc)2 (22.40 mg, 0.10 mmol) were placed in a 250 mL round-bottom flask, and toluene (30.0 mL) was added. The temperature was adjusted to 100 °C, and the mixture was stirred for 12 h under nitrogen protection and allowed to react overnight.

[0075] (2) After cooling to room temperature, add water (80.00 mL) and chloroform (200.00 mL). Allow to stand for phase separation, separate and wash the organic layer, and dry with anhydrous MgSO4 to obtain the crude product. Prepare an eluent: petroleum ether / dichloromethane (v1 / v2 = 20 / 1), and purify by silica gel column chromatography to obtain the solid product TPATBS.

[0076] The reaction route is as follows:

[0077]

[0078] 2. Characterization tests of TPATBS

[0079] (1) Nuclear magnetic resonance and mass spectrometry detection

[0080] The proton NMR results of TPATBS are as follows: Figure 1 As shown, the chemical structure of TPATBS was determined, and the specific NMR data are as follows:

[0081] 4,4'-(Benzo[c][1,2,5]selenadiazole-4,7-diylbis(thiophene-5,2-diyl))bis(N,N-diphenylaniline)(TPABT-Se):1H NMR (400MHz, CDCl3) δ (ppm) 8.01 (d, J = 3.9Hz, 2H), 7.79 (s, 2H), 7.58-7.56 (m, 4H), 7.32-7.28 (m, 10H), 7.15-7.04 (m, 16H).

[0082] Sample solution was added to the mass spectrometer sample plate and allowed to dry. Analysis was performed using a high-performance liquid chromatography-electrospray ionization trap / time-of-flight tandem mass spectrometer. Results are as follows: Figure 2 As shown, the results indicate that the theoretical value of the molecular weight of TPATBS is 834.045, while the actual value is 834.190, showing that the theoretical value is consistent with the actual value.

[0083] (2) Measurement of UV absorption and fluorescence emission spectra of TPATBS

[0084] The ultraviolet absorption and fluorescence emission spectra of TPATBS were measured using a UV-Vis spectrophotometer and a fluorescence spectrometer. The results are as follows: Figure 3 As shown, its absorption and emission peaks are concentrated at 560 nm and 688 nm, respectively, exhibiting near-infrared fluorescence emission characteristics.

[0085] (3) Determination of AIE properties of TPATBS molecules

[0086] TPATBS was dissolved in a mixture of a good solvent and a bad solvent, with chloroform as the good solvent and n-hexane as the bad solvent. Its concentration in chloroform / n-hexane mixtures at different ratios was determined. H =0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%, f H The fluorescence intensity distribution of TPATBS molecules (where n-hexane accounts for a certain percentage in the mixed solution) was determined by fluorescence spectroscopy. The changes in fluorescence emission intensity of TPATBS molecules under different ratios of chloroform / n-hexane were detected using a fluorescence spectrometer. Specific results are shown below. Figure 4 As shown, TPATBS molecules exhibit weak fluorescence emission in pure chloroform solution. Figure 4 (a) As the undesirable solvent n-hexane is gradually added, the fluorescence emission gradually increases. Figure 4 (b) indicates that the TPATBS molecule has excellent AIE performance.

[0087] Example 2: Preparation and Characterization of TPATBS NPs

[0088] 1. TPATBS NPs were prepared using a nano-coprecipitation method (reaction process as follows). Figure 5 (As shown)

[0089] First, 5.0 mg of TPATBS compound and 500 mg of Pluronic F127 were weighed and dissolved in a round-bottom flask containing 20 mL of THF. The solution was then sonicated at 250 W for 30 min. The organic solvent THF was removed by rotary evaporation, and 30 mL of deionized water was added for further sonication. The solution was then placed in a dialysis bag (3500 Da) and dialyzed in deionized water for 48 h. The TPATBS NPs aqueous solution was filtered using a 200 μM pore size membrane. The resulting TPATBS NPs aqueous solution was stored at 4 °C for subsequent experiments.

[0090] 2. Characterization of TPATBS NPs

[0091] (1) Measurement of optical properties of TPATBS NPs

[0092] The absorption and emission characteristics of TPATBS NPs were determined using a UV-Vis spectrophotometer and a fluorescence spectrometer. The results are as follows: Figure 6 As shown, the maximum absorption peak and emission peak of TPATBS NPs reach 570 nm and 712 nm, respectively, which are significantly redshifted compared to TPATBS molecules. This makes TPATBS NPs a potential application in near-infrared fluorescence imaging.

[0093] (2) Determination of particle size and stability of TPATBS NPs

[0094] The morphology, particle size, and stability of the prepared nanoparticles were analyzed using transmission electron microscopy (TEM) and dynamic light scattering (DLS). The results are as follows: Figure 7 As shown in (ac), these nanoparticles exhibit a uniform spherical morphology. TPATBS NPs possess a uniform size of 120 nm, which is beneficial for their accumulation at tumor sites via the EPR effect. Their zeta potentials are -12.15 eV, and their negative surface charge contributes to their stability in biological systems.

[0095] To further understand the cyclic stability of nanoparticles in biological systems, the changes in hydrated particle size of TPATBS NPs were determined after placement in DMEM, FBS, PBS, and ultrapure water at different time intervals (1, 7, and 14 days). The results are as follows: Figure 8 As shown, the hydrated particle size of the nanoparticles did not change significantly in different solvents.

[0096] Test Example 1: Detection of ROS generation capacity and photothermal effect of TPATBS NPs

[0097] 1. ROS determination

[0098] Mix 100 μL of 1 mM DCFH-DA stock solution with 400 μL of 0.01 M NaOH aqueous solution, and incubate at room temperature for 30 min to convert DCFH-DA to DCFH. Add 19.5 μL of PBS solution to bring the final concentration to 5 mM. Then add TPATBS NPs and RB, and irradiate with white light (light intensity 60 mW / cm²). 2 After different time intervals, the fluorescence emission intensity of the mixed solution at 525 nm (EX: 485 nm) was measured.

[0099] The results are as follows Figure 9 As shown, this demonstrates that TPATBS NPs have excellent ROS generation capabilities.

[0100] 2. Photothermal effect detection

[0101] The concentrations of TPATBS NPs in aqueous solutions of different concentrations (0, 22.5 μmol / L, 45.0 μmol / L, 90.0 μmol / L, and 180 μmol / L) at 800 mW / cm² were determined. 2 Photothermal effect under laser irradiation, and TPATBS NPs aqueous solution with a concentration of 90 μM / L at different optical power densities (0.0 W / cm²). 2 0.4W / cm 20.6W / cm 2 0.8W / cm 2 1.0W / cm 2 1.2W / cm 2 The photothermal effect under laser irradiation.

[0102] The results are as follows Figure 10 As shown, the photothermal conversion effect is positively correlated with the concentration of the nanoparticle aqueous solution and the optical power density. A 635nm laser irradiation (0.8W / cm²) was selected. 2 The temperature of the TPATBS NPs aqueous solution rapidly increased within 2 minutes, mainly attributed to its twisted configuration and structurally rich rotors, resulting in intense intramolecular motion within the aggregates. After 5 minutes of laser irradiation, the maximum temperature of the TPATBS NPs reached 67.5 °C. The photothermal conversion efficiency of TPATBS NPs was as high as 41.47%. Furthermore, the maximum heating temperature of the TPATBS NPs sample remained almost unchanged during 6 heating-cooling cycles, demonstrating its excellent photothermal stability.

[0103] The TPATBS NPs of this invention exhibit excellent ROS generation capacity and photothermal conversion effect under light irradiation, enabling synergistic tumor phototherapy using photothermal phototherapy (PDT) and photothermal phototherapy (PTT). PTT, through local laser irradiation of the tumor site, raises the temperature at the tumor site, promoting blood circulation and further alleviating the hypoxia problem in the tumor microenvironment, thus enhancing the effect of PDT. Compared with existing technologies, the single-molecule TPATBS NPs designed and prepared in this invention have advantages such as simple synthesis, convenient application, and significant anti-tumor effects, and can significantly improve the efficacy of tumor phototherapy.

[0104] Test Example 2: Cytotoxicity Assessment of TPATBS NPs

[0105] The cytotoxicity of TPATBS NPs in the MDA-MB-231 cell line was evaluated using the CCK-8 assay. The specific testing steps are as follows:

[0106] (1) With 5×10 per hole 3 Cell density was determined by seeding MDA-MB-231 cell lines into 96-well plates.

[0107] (2) After culturing for 24 hours, discard the culture medium and add fresh DMEM medium containing different concentrations of TPATBS NPs (0, 10 μM, 20 μM, 40 μM, 80 μM).

[0108] (3) Continue culturing for 12h, 24h, 36h and 48h respectively, then remove the culture medium and wash twice with PBS. Add 10μL of CCK-8 reagent and 90μL of colorless DMEM culture medium to each well, incubate for 45min, and then use an ELISA reader to measure absorbance at 450nm wavelength to assess cell viability.

[0109] The results are as follows Figure 11 As shown, the cell viability of MDA-MB-231 cells after co-incubation with TPATBS NPs for 12h, 24h, 36h, and 48h did not decrease significantly, indicating that low concentrations of TPATBS NPs are not cytotoxic under dark conditions and can be applied to biological systems.

[0110] Test Example 3: Detection of in vivo and in vitro fluorescence imaging capabilities

[0111] To evaluate the time-dependent uptake and fluorescence imaging capabilities of TPATBS NPs, we co-incubated TPATBS NPs with MDA-MB-231 cells for different times (2h, 4h, 6h, 8h), and detected the fluorescence signal intensity of different treatment groups using laser confocal scanning microscopy (CLSM). The results are as follows: Figure 12 As shown, this indicates that TPATBS NPs are taken up by MDA-MB-231 cells in a time-dependent manner.

[0112] A subcutaneous tumor model of MDA-MB-231 was constructed in female BALB / c nude mice (model construction reference 1. Chen, X.; Mendes, BB; Zhuang, Y.; Conniot, J.; Mercado Argandona, S.; Melle, F.; Sousa, DP; Perl, D.; Chivu, A.; Patra, HK; et al. A Fluorinated BODIPY-Based Zirconium Metal-Organic Framework for In Vivo Enhanced Photodynamic Therapy. J Am Chem Soc 2024, 146(2), 1644-1656. DOI: 10.1021 / jacs.3c12416 From NLM.). When the mice reached 5 weeks of age, 5 × 10⁻⁶ mcg of the upper right leg was injected subcutaneously. 6 One MDA-MB-231 cell was used until the tumor grew to 100 mm. 3At that time, 100 μL and 40 μM of TPATBS NPs aqueous solution were injected into the mice via tail vein and subcutaneous injection, respectively. The fluorescence intensity of the tumor sites in mice was measured at different time points (0h, 1h, 3h, 6h, 12h, 24h, 36h, 2day…14day, 15day). The results are as follows: Figure 13 As shown, TPATBS NPs have good fluorescence imaging capabilities at the animal level and have the potential for long-term tumor monitoring, up to 15 days, which is far superior to conventional fluorescence imaging agents.

[0113] The TPATBS NPs of this invention possess the advantage of ultra-long-term fluorescence imaging, indicating their excellent stability and resistance to signal attenuation due to continuous photoexcitation. They also avoid interference from frequent probe labeling or reagent replenishment, enabling continuous dynamic monitoring of biological processes, such as cell division, growth, tumor migration, and drug metabolism. Furthermore, they effectively reduce the dosage required for biological administration, significantly decreasing biotoxicity and improving biosafety.

[0114] Example 6: Evaluation of in vivo photothermal effect and phototherapy effect

[0115] A subcutaneous tumor model of MDA-MB-231 was constructed in BALB / c nude mice, and the tumor was allowed to grow to 100 mm. 3 The mice were divided into four groups: PBS, PBS+Laser, TPATBS NPs, and TPATBS NPs+Laser, with six mice in each group. 100 μL of either TPATBS NPs or PBS was injected into the tumor site. One hour later, the PBS+Laser and TPATBS NPs+Laser groups were irradiated with a 630nm laser, respectively. Photothermal imaging was performed at time points of 0 min, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, and 10 min. The results are as follows: Figure 14 As shown, the TPATBS NPs+Laser group exhibited good photothermal imaging effects. After treatment, tumor volume and body weight were measured in mice, and the results are as follows. Figure 15 As shown in Figure a, the tumor volume of mice treated with TPATBS NPs and laser irradiation was significantly reduced, and the tumors were completely ablated after four treatments, with no significant change in mouse body weight, indicating that TPATBS NPs have low biotoxicity (see Figure a). Figure 15 b). Subsequently, we stained the heart, liver, spleen, lungs, and kidneys of mice in different treatment groups with hematoxylin and eosin (HE), and the results are as follows. Figure 15As shown in Figure c, there was no obvious damage to the organs of mice in both the experimental and control groups, further demonstrating that TPATBS NPs did not cause significant toxic side effects to mice under laser irradiation for tumor phototherapy, indicating that the TPATBS NPs of the present invention have good biosafety.

[0116] The phototherapy agent TPATBS NPs of this invention is synthesized using Suzuki coupling reaction and nano-self-assembly technology. These nanoparticles exhibit good biocompatibility, excellent fluorescence imaging properties, high ROS generation capacity, and significant photothermal conversion effect. Furthermore, the TPATBS NPs provided by this invention have a strong inhibitory effect on tumor cell growth, demonstrating good anti-tumor efficacy. Experimental results show that this phototherapy agent—TPATBS NPs—can be applied to long-term tumor monitoring and synergistic phototherapy using photodynamic therapy (PDT) and photothermal therapy (PTT), achieving multimodal imaging-guided tumor phototherapy and providing a new approach for the integrated diagnostic and therapeutic application of AIE (autoimmune endoscopic interventional) agents.

[0117] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A near-infrared AIE phototherapy agent having a DAD structure, comprising a compound with the structural formula shown in Formula I, or a pharmaceutically acceptable salt thereof, a solvate thereof, and a tautomer thereof; in, R is one or more of O, S, and Se; M is n Where X represents O, S, or Se, n = 0, 1, 2, or 3, and the dashed line represents the connection point.

2. The near-infrared AIE phototherapy agent according to claim 1, characterized in that, n is 1 or 2.

3. The near-infrared AIE phototherapy agent according to claim 1 or 2, characterized in that, It includes compounds represented by the following structural formulas, or pharmaceutically acceptable salts thereof, or solvates thereof, or tautomers thereof:

4. The near-infrared AIE phototherapy agent according to any one of claims 1 to 3, characterized in that, It is a nanotherapeutic agent, optionally formed by coating a compound as shown in Formula I and an amphiphilic polymer coating agent via a nano-co-precipitation method; Optionally, the external structure of the near-infrared AIE phototherapy agent is a DSPEG-mPEG2000 or Pluronic F127 amphiphilic polymer coating agent, and the core structure is a aggregation-induced emission material TPATBS that generates heat under laser excitation, forming an aggregation-induced emission phototherapy agent TPATBS NPs.

5. A method for preparing a near-infrared AIE phototherapy agent according to any one of claims 1 to 4, characterized in that, The process includes the following steps: In a solvent, under the action of an alkaline substance and Pd(OAc)₂, the compounds shown in formulas A and B are converted into the compound shown in formula I: The M and R in this case are the same as those in compound I.

6. The preparation method according to claim 5, characterized in that, The solvent is selected from one or more of toluene, acetonitrile, dimethyl sulfoxide, and dimethylformamide; And / or, the alkaline substance is selected from one or more of potassium carbonate, sodium carbonate, and cesium carbonate; And / or, the reaction is carried out in an inert gas atmosphere, optionally with nitrogen; And / or, the reaction temperature is 40–120°C, optionally 80–120°C, optionally 100–110°C; And / or, the reaction time is 10 to 18 hours, optionally 12 to 18 hours; And / or, the molar ratio of the compounds shown in Formula A and Formula B is 1:0.9 to 1.5, optionally 1:1 to 1.5, optionally 1:1 to 1.

2.

7. The preparation method according to claim 5 or 6, characterized in that, Nanoparticles were prepared from the compound shown in Formula I: The compound shown in Formula I and the amphiphilic polymer were dissolved in an organic solvent, and nanoparticles were obtained by sonication, dialysis, and filtration. Optionally, the amphiphilic polymer is Pluronic F127 or DSPEG-mPEG-2000; Optionally, the mass ratio of the compound shown in Formula I to the amphiphilic polymer is 1:30 to 1:150, optionally 1:30 to 1:100, optionally 1:100 to 150, optionally 1:

100.

8. The preparation method according to claim 7, characterized in that, The organic solvent is selected from one or more of THF, ACN, DMSO, and DMF; Optionally, the diameter of the nanoparticles is 100–150 nm; Optionally, the ultrasound time is 1–50 min, or optionally 30–40 min; Optionally, the ultrasonic power is 20-300W, and optionally 250W; Optionally, dialysis is performed in deionized water, and the deionized water is changed every 6 to 10 hours. Optionally, dialysis is performed for 24 to 56 hours, or 48 to 56 hours. Optionally, the pore size of the filter is 150–200 μM, optionally 200 μM.

9. The application of a near-infrared AIE phototherapy agent according to any one of claims 1 to 4, or a near-infrared AIE phototherapy agent prepared by any one of claims 5 to 8, in the preparation of tumor diagnosis, treatment, and integrated diagnostic and therapeutic or anti-tumor products, wherein the product may optionally be a long-term tumor monitoring probe, a multimodal imaging contrast agent, and a superficial tumor phototherapy agent.

10. The application according to claim 9, characterized in that, Near-infrared AIE phototherapy agent is used under 635nm laser irradiation. Optionally, the near-infrared AIE phototherapy agent can reach a photothermal temperature of 65-69°C under 635nm laser irradiation intensity, and optionally 67.5-69°C; Optionally, the imaging capability of the near-infrared AIE phototherapy agent is maintained for 10 to 20 days, or optionally 12 to 15 days.

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