Fluorescent compounds for cancer diagnosis and treatment in cooperation with photodynamic and photothermal

Abstract

The invention discloses an aggregation-induced emission fluorescent compound with near infrared (NIR) luminescence. The invention also shows the application of combining photodynamic therapy and photothermal therapy in image-guided optical diagnosis and treatment. In-vitro and in-vivo verification further confirms that the system shows excellent fluorescence-guided optical diagnosis and treatment capability and can effectively eliminate tumors.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This invention is a Chinese national phase application of international application No. PCT / CN2023 / 122882 filed on September 28, 2023, which claims priority to and the benefits of U.S. Provisional Patent Application No. 63 / 416,936, filed on October 17, 2022. The entire contents of each of the aforementioned applications are incorporated herein by reference. Technical Field

[0003] The present invention relates to an aggregation-induced emission fluorescent compound that emits near-infrared (NIR) light, and applies the compound to image-guided optical diagnosis and treatment. Background Art

[0004] Cancer is one of the most serious health problems in the world, causing extremely high mortality rates. Although a variety of interventions such as chemotherapy, radiotherapy and surgery have been used to treat cancer, their serious side effects and unsatisfactory treatment outcomes are still far from satisfactory. Therefore, the development of more effective cancer therapies is necessary but challenging. Fluorescence-guided optical diagnostics and treatment technology uses light irradiation to simultaneously diagnose and treat cancer, and has become a promising tool due to its controllability, non-invasiveness and simultaneous treatment process. However, robust phototherapy systems that can simultaneously have diagnostic and therapeutic capabilities are extremely scarce because it is very difficult to deal with complex energy attenuation pathways during the excitation process.

[0005] It is well known that during photoexcitation, a typical radiative pathway for molecules to process and receive energy after being excited is fluorescence (FL). Another pathway is non-radiative dissipation, which can be used for photothermal therapy (PTT). In addition, some photosensitizers can achieve photodynamic therapy (PDT) by generating reactive oxygen species (ROS) through intersystem crossing (ISC).

[0006] In the field of optical diagnosis and treatment, photothermal therapy (PTT) and photodynamic therapy (PDT) are two promising treatment methods with extremely low drug resistance, non-invasiveness, and high sensitivity. However, using PDT or PTT alone is not always sufficient to eliminate tumors because the hypoxic microenvironment of tumor tissue may limit the efficiency of PDT, and temperature increase alone is not sufficient to eliminate lesions. Therefore, the development of advanced materials to integrate PDT and PTT for synergistic cancer treatment is extremely necessary. Summary of the Invention

[0007] The present invention provides an aggregation-induced luminescent fluorescent compound that emits near-infrared (NIR) light. The present invention also demonstrates its application in image-guided optical therapy, combining photodynamic therapy and photothermal therapy. In vitro and in vivo validation further confirms that this system demonstrates excellent fluorescence-guided optical therapy capabilities and can effectively eliminate tumors.

[0008] In one embodiment, the present invention discloses a fluorescent compound. The fluorescent compound exhibits aggregation-induced emission characteristics and has the following main structural formula:

[0009]

[0010] wherein each of R1, R2 and R3 is independently selected from the following: linear, branched, cyclic alkyl, alkylphenyl, alkylthienyl and other alkyl aromatic groups having 2 to 40 carbon atoms, and wherein one or more non-adjacent carbon atoms are optionally replaced by –O–, –S–, –C(O)–, –C(O–)–O–, –O–C(O)–, –O–C(O)–O– or –C≡C–, and wherein one or more hydrogen atoms are optionally replaced by fluorine (F), chlorine (Cl), bromine (Br), iodine (I) or cyanide (CN), or represents an aromatic, heteroaromatic, aryloxy, heteroaryloxy, aromatic carbonyl, heteroaromatic carbonyl, aromatic carbonyloxy, heteroaromatic carbonyloxy, aryloxycarbonyl or heteroaryloxycarbonyl group having a ring of 4 to 30 atoms, and these ring molecules are unsubstituted or substituted by one or more non-aromatic groups; and

[0011] Wherein each of R4 and R5 is independently selected from the following combinations:

[0012]

[0013] In another embodiment, the present invention discloses a therapeutic diagnostic agent, which includes the aforementioned fluorescent compound in the form of nanoparticles, and a polymer matrix encapsulating the aforementioned fluorescent compound.

[0014] In another embodiment, the present invention discloses a method for killing cancer cells, comprising:

[0015] directing the aforementioned therapeutic diagnostic agent to contact the target cancer cells;

[0016] When the theranostic agent contacts the target cancer cells, imaging the target cancer cells using an imaging method selected from the group consisting of fluorescence microscopy, bioluminescence imaging, and confocal laser scanning microscopy; and

[0017] The target cancer cells are placed under light irradiation, and the aforementioned therapeutic diagnostic agent contacts the target cancer cells to kill the target cancer cells.

[0018] In yet another embodiment, the present invention discloses a method for stopping or inhibiting tumor growth in a mammal, comprising:

[0019] administering the aforementioned theranostic agent to a mammal;

[0020] directing the aforementioned therapeutic diagnostic agent to the tumor site for contact;

[0021] After the tumor site is contacted with the aforementioned theranostic agent, locating the tumor site using an imaging method; and

[0022] When the aforementioned theranostic agent is present at the tumor site, light is irradiated to the tumor site to stop or inhibit tumor growth.

[0023] The above description is only a summary of the technical solution of the present invention. The following provides a complete embodiment of the present invention in conjunction with the accompanying drawings to make it easier for those skilled in the art to understand the operation process and its objectives, features and advantages of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present invention will be fully understood from the following detailed description of the complete embodiments with reference to the accompanying drawings, in which:

[0025] Figure 1 The synthetic routes of TPA-TBT and TPE-TBT are shown.

[0026] Figure 2 The molecular structures of TPA-TBT and TPE-TBT are shown.

[0027] Figure 3a Shows the normalized absorption spectra of TPA-TBT and TPE-TBT (10 μM) in tetrahydrofuran (THF) solution. Figure 3b Normalized fluorescence spectra are shown. Figure 3c The graph shows the relative photoluminescence (PL) intensity (I / I0) of TPA-TBT at 830 nm and the relative photoluminescence (PL) intensity (I / I0) of TPE-TBT at 750 nm in different water components. Figure 3d The fluorescence quantum yields of the compounds in THF solution, nanoparticles (NPs), and solid state are shown. Figure 3e Showing the ROS generation ability of TPA-TBT and TPE-TBT nanoparticles under white light irradiation. Figure 3f Displayed at 660nm laser (1W / cm 2 ) photothermal properties of nanoparticles under irradiation.

[0028] Figure 4 The dynamic light scattering (DLS) spectrum of TPA-TBT nanoparticles is shown.

[0029] Figure 5Shown are the dynamic light scattering (DLS) spectra of TPE-TBT nanoparticles.

[0030] The relative survival rates of 293T, Hela and 4T1 cells after treatment with different concentrations of TPA-TBT nanoparticles. Figure 6a is 0 hours; Figure 6b for 24 hours, and then white light (11mW / cm 2 ) for 10 minutes. Figure 6c Flow cytometric analysis of 4T1 cells after various treatments, including untreated cells (control), cells treated with TPA-TBT nanoparticles, cells treated with a 660 nm laser, and cells treated with both nanoparticles and lasers. Laser power: 1 W / cm 2 .

[0031] Figure 7 CLSM imaging of ROS inside 4T1 cells using H2DCF-DA staining under different experimental conditions, including control group, TPA-TBT nanoparticles, TPA-TBT nanoparticles + white light (white light, 11 mW / cm 2 , 10 minutes; excitation wavelength: 488 nm; filter: 500-550 nm). DAPI is a nuclear dye (excitation wavelength: 405 nm; filter: 460-500 nm).

[0032] Figure 8a Shown are time-dependent near-infrared fluorescence images of 4T1 tumor-bearing mice after TPA-TBT nanoparticle injection. Figure 8b The quantitative fluorescence intensity of tumor tissue after different monitoring times is shown. Figure 8c The results show that the laser irradiation at 660 nm (0.5 W / cm 2 ) Infrared thermal images of 4T1 tumor mice at different times after nanoparticle injection. Figure 8d The results show that the laser irradiation at 660 nm (0.5 W / cm 2 ) for 10 minutes after intravenous injection of TPA-TBT nanoparticles and PBS in mice with 4T1 tumors. Figure 8e Figure 3. Relative tumor volume changes after various treatments. Inset: Representative tumor images of the four groups after 14 days of treatment (from left to right: PBS, TPA-TBT nanoparticles, laser, TPA-TBT nanoparticles + laser). Figure 8f Photos of mice with tumors in different groups are shown. Figure 8g H&E and TUNEL staining analyses of tumor tissues after various treatments are shown.

[0033] Figure 9Shown are representative near-infrared-I (NIR-I) fluorescence images of ex vivo organs harvested from 4T1 tumor-bearing mice 12 hours after injection of TPA-TBT nanoparticles and quantitative analysis of their mean fluorescence intensity.

[0034] Figure 10 Shown are in vivo photothermal images of 4T1 tumor-bearing mice after administration of PBS or TPA-TBT nanoparticles (1.0 mg / mL, 200 μL) under 660 nm near-infrared laser irradiation.

[0035] Figure 11 Tumor images of the four groups after 14 days of treatment are shown (from left to right: PBS, TPA-TBT nanoparticles, laser, TPA-TBT nanoparticles + laser).

[0036] Figure 12 The following table shows the body weight of mice within 14 days after different treatments. No obvious side effects were observed in any group.

[0037] Figure 13 Shows the blood routine data of BALB / c female mice after 7 or 14 days of treatment with PBS or TPA-TBT nanoparticles, including white blood cells (WBC), red blood cells (RBC), platelets (PLT), lymphocytes (Lymph), hemoglobin (HGB), and calcitonin (PCT). (n=3 per group)

[0038] Figure 14 Figure 3 shows the blood biochemical data of BALB / c female mice treated with PBS or TPA-TBT nanoparticles for 7 or 14 days, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), creatinine (CR), total cholesterol (TCH), and urea (UREA). (n=3 per group)

[0039] Figure 15 H&E staining images of major organs (including heart, liver, spleen, lung, and kidney) of BALB / c female mice treated with PBS or TPA-TBT nanoparticles for 7 or 14 days. Scale bar: 100 μm. DETAILED DESCRIPTION

[0040] definition

[0041] In this application, when an element or component is referred to as being included in and / or selected from a list of enumerated elements or components, it is understood that the element or component can be any of the enumerated elements or components, or the element or component can be selected from a group consisting of two or more of the enumerated elements or components. Furthermore, it is understood that the elements, compound group properties, apparatuses, or methods described herein may be combined in various ways, whether explicitly or implicitly herein, without departing from the spirit and scope of the present teachings.

[0042] Use of the terms "including," "comprising," "having," or "having" are generally accepted to be open ended and non-limiting unless expressly stated otherwise.

[0043] The singular is used herein to include the plural (and vice versa) unless otherwise expressly stated. Furthermore, when the term "about" is used before a quantitative value, this patent also includes the specific quantitative value itself, unless otherwise expressly stated. When the term "about" is used herein, it means within a range of ±10% from the standard value, unless otherwise stated or inferred.

[0044] The order in which the steps are performed is not critical, as long as the guidelines in the patent remain valid. In addition, two or more actions may be performed simultaneously.

[0045] “λ ex ” here refers to the excitation wavelength.

[0046] The term "aggregation-induced luminescence quenching" or "ACQ" as used herein refers to the phenomenon that aggregation of π-conjugated fluorophores significantly reduces the fluorescence intensity of the fluorophore. The formation of aggregates is believed to "quench" the luminescence of the fluorophore.

[0047] The term "aggregation-induced emission" or "AIE" as used herein refers to a phenomenon in which compounds exhibit significantly enhanced luminescence when aggregated in the amorphous or crystalline (solid state) but exhibit weak or almost no luminescence in dilute solution.

[0048] "Luminescence intensity" as used herein refers to the amount of fluorescence / phosphorescence typically measured using a fluorescence spectrometer or fluorescence microscope; "fluorophore" or "fluorescence source" as used herein refers to a molecule that exhibits fluorescence; "luminescence source" or "luminophore" as used herein refers to a molecule that exhibits luminescence;

[0049] Here, "AIEgen" refers to a molecular cluster that exhibits AIE properties.

[0050] As used herein, a "donor" material refers to an organic material, such as an organic nanoparticle material, in which holes serve as the primary current or charge carriers.

[0051] As used herein, an "acceptor" material refers to an organic material, such as an organic nanoparticle material, in which electrons are the primary current or valence charge carriers.

[0052] As used herein, a "theranostic agent" refers to an organic material, such as an organic nanoparticle material, that possesses both diagnostic and therapeutic capabilities.

[0053] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0054] Fluorescent compounds and complexes

[0055] Among various phototheranostics, fluorescent nanoparticles (NPs) are particularly popular due to their high sensitivity and temporal resolution of fluorescence imaging, real-time and in situ labeling for photodynamic therapy (PDT), and unique enhanced permeability and retention (EPR) effect. To meet the requirements of ideal cancer phototheranostics, fluorescent nanoparticles must possess several properties, including sufficiently strong near-infrared (NIR) luminescence (>650nm); efficient reactive oxygen species (ROS) generation by the fluorescent components within the nanoparticles; strong resistance to photobleaching; negligible cytotoxicity and in vivo toxicity; and suitable nanoparticle size and surface chemistry that allow for a significant EPR effect.

[0056] Aggregation-induced emission materials (AIEgens) have recently emerged as alternative fluorescent materials for constructing fluorescent nanoparticles (NPs), perfectly solving the challenge of aggregation-induced luminescence quenching (ACQ) and showing low in vivo toxicity. AIEgens generally do not emit light in solution because the excited state energy is consumed through non-radiative relaxation of intramolecular motion. When aggregated, the relaxation process from the lowest excited singlet state (S1) to the ground state (S0) is greatly restricted due to steric effects, causing the energy of S1 to be transferred to S0 through the fluorescence pathway. This unusual property makes AIEgens very suitable for the fabrication of fluorescent nanoparticles with ultrahigh brightness and photobleaching threshold.

[0057] The present invention constructs a fluorescent compound that exhibits aggregation-induced emission (AIE) and near-infrared luminescence. Near-infrared (NIR) fluorescence imaging has attracted widespread attention due to its lower interference from tissue autoluminescence and deeper tissue penetration compared to visible light. The compound can be present in the form of nanoparticles.

[0058] The present invention also provides compositions comprising a fluorescent compound and a polymer matrix encapsulating the fluorescent compound. These compositions may also be in the form of nanoparticles. Compositions comprising nanoparticles of the fluorescent compound and a polymer matrix are also referred to herein as "theranostic agents." Encapsulating the fluorescent compound in a polymer matrix can improve the microenvironment within the particle, thereby increasing its fluorescence intensity and reactive oxygen species (ROS) generation capacity in vivo.

[0059] In one embodiment, the present invention provides a fluorescent compound exhibiting aggregation-induced emission characteristics, wherein the fluorescent compound has the following main structural formula:

[0060]

[0061] wherein each of R1, R2 and R3 is independently selected from the following: linear, branched, cyclic alkyl, alkylphenyl, alkylthienyl and other alkyl aromatic groups having 2 to 40 carbon atoms, and wherein one or more non-adjacent carbon atoms are optionally replaced by –O–, –S–, –C(O)–, –C(O–)–O–, –O–C(O)–, –O–C(O)–O– or –C≡C–, and wherein one or more hydrogen atoms are optionally replaced by fluorine (F), chlorine (Cl), bromine (Br), iodine (I) or cyanide (CN), or represents an aromatic, heteroaromatic, aryloxy, heteroaryloxy, aromatic carbonyl, heteroaromatic carbonyl, aromatic carbonyloxy, heteroaromatic carbonyloxy, aryloxycarbonyl or heteroaryloxycarbonyl group having a ring of 4 to 30 atoms, and these ring molecules are unsubstituted or substituted by one or more non-aromatic groups; and

[0062] Wherein each of R4 and R5 is independently selected from the following combinations:

[0063]

[0064] In this embodiment, R1, R2, and R3 may be branched alkyl groups having 2 to 40 carbon atoms.

[0065] Preferably, the fluorescent compound is selected from the following combinations:

[0066]

[0067] In another embodiment, the present invention provides a fluorescent nanoparticle composition comprising a fluorescent compound having aggregation-induced emission properties and a polymer matrix, wherein the polymer matrix encapsulates the fluorescent compound, and the fluorescent compound has the following main structural formula:

[0068]

[0069] wherein each of R1, R2 and R3 is independently selected from the following: linear, branched, cyclic alkyl, alkylphenyl, alkylthienyl and other alkyl aromatic groups having 2 to 40 carbon atoms, and wherein one or more non-adjacent carbon atoms are optionally replaced by –O–, –S–, –C(O)–, –C(O–)–O–, –O–C(O)–, –O–C(O)–O– or –C≡C–, and wherein one or more hydrogen atoms are optionally replaced by fluorine (F), chlorine (Cl), bromine (Br), iodine (I) or cyanide (CN), or represents an aromatic, heteroaromatic, aryloxy, heteroaryloxy, aromatic carbonyl, heteroaromatic carbonyl, aromatic carbonyloxy, heteroaromatic carbonyloxy, aryloxycarbonyl or heteroaryloxycarbonyl group having a ring of 4 to 30 atoms, and these ring molecules are unsubstituted or substituted by one or more non-aromatic groups; and

[0070] Wherein each of R4 and R5 is independently selected from the following combinations:

[0071]

[0072] In this embodiment, R1, R2, and R3 may be branched alkyl groups having 2 to 40 carbon atoms.

[0073] Preferably, the fluorescent compound is selected from the following combinations:

[0074]

[0075] Figure 1 An exemplary reaction scheme for the preparation of TPA-TBT and TPE-TBT compounds is presented.

[0076] As mentioned above, during photoexcitation, molecules are excited and then processed to receive energy. One typical radiative pathway is fluorescence emission (FL). Another pathway is nonradiative dissipation, which can achieve a photothermal effect, which can be used for photothermal therapy (PTT). In addition, some photosensitizers can achieve photodynamic therapy (PDT) by generating reactive oxygen species (ROS) through intersystem crossing (ISC). Since the total energy is fixed, a balance should exist that can flexibly utilize all energy relaxation processes to achieve the most effective image-guided phototherapy using a single molecule.

[0077] In a preferred embodiment of the present invention, an integrated AIE system integrating photodynamic therapy (PDT) and photothermal therapy (PTT) is provided for image-guided optical diagnosis and treatment. By rationally comparing the donor part, the strong electron donor group triphenylamine (TPA) was selected, and combined with thiadiazole benzotriazine (TBT) as an acceptor, an AIEgen TPA-TBT with high quantum yield and near-infrared luminescence characteristics was realized. The prepared TPA-TBT nanoparticles (TPA-TBT NPs) exhibited excellent biocompatibility, AIE properties and aggregation ability in solid tumors. It was found that TPA-TBT NPs have long-wavelength luminescence, high fluorescence quantum yield, excellent photothermal conversion efficiency and significant reactive oxygen species (ROS) generation ability.

[0078] Cancer diagnosis and / or cancer treatment

[0079] The theranostic agents described herein have potential benefits in cancer diagnosis and optical therapy applications, particularly in near-infrared image-guided cancer surgery combined with photodynamic therapy (PDT) and photothermal therapy (PTT). Image-guided cancer surgery using near-infrared fluorescence has been demonstrated to be feasible in clinical cancer surgery and has great potential to improve the success rate of cancer surgery. The theranostic agents described herein can serve as efficient near-infrared fluorescent probes that meet the necessary requirements for image-guided cancer surgery.

[0080] As described herein, TPA-TBT and TPE-TBT were prepared into nanoparticles (NPs) by nanoprecipitation and DSPE-PEG2000 was used as the coating matrix. The hydrodynamic diameters of the two nanoparticles were determined by dynamic light scattering (DLS). The results showed that TPA-TBT and TPE-TBT nanoparticles were uniformly dispersed in water with diameters of approximately 37 nm and 45 nm, respectively (Figure 2). Figure 4 、 Figure 5 ).

[0081] The fluorescence quantum yields (QY) of the two compounds in solid state, nanoparticles (NPs), tetrahydrofuran (THF) and other solvents were also measured (e.g. Figure 3d The results showed that TPA-TBT nanoparticles exhibited a high absolute quantum yield of 7%, while TPE-TBT nanoparticles achieved an even higher quantum yield of 28%. The quantum yield of TPA-TBT in the solid state was 13%, higher than its quantum yield (5%) in nanoparticles and THF solution. In contrast, TPE-TBT exhibited a significant quantum yield in solution, demonstrating that TPE-TBT exhibits a more pronounced TICT effect and active radioactive decay.

[0082] To further explore the phototherapeutic potential of these AIEgens, the ROS generation efficiency and photothermal conversion efficiency (PCE) were studied. A common ROS indicator, dichlorofluorescein diacetate (H2DCF-DA), whose fluorescence can be sensitively activated by ROS, was used to indicate the overall ROS generation. Figure 3e It can be seen that in the presence of TPA-TBT nanoparticles, the fluorescence intensity of H2DCF-DA rapidly increased, eventually reaching approximately 210 times the initial intensity after 5 minutes of white light irradiation, indicating a higher ROS production efficiency than that of TPE-TBT nanoparticles.

[0083] In addition, TPA-TBT nanoparticles exhibited better photothermal performance. Figure 3f As shown, after 350 seconds of 660nm laser irradiation, TPA-TBT nanoparticles reached a temperature of approximately 57°C, with a photothermal conversion efficiency (PCE) of 44%. In contrast, the PCE of TPE-TBT nanoparticles was only 30%. Taking all factors into consideration, TPA-TBT nanoparticles exhibit longer fluorescence wavelengths, higher reactive oxygen species generation efficiency, and better photothermal efficiency. Therefore, TPA-TBT nanoparticles hold great promise for application in optical diagnostics and therapeutics for synergistic cancer treatment.

[0084] According to the aforementioned embodiment, the present invention provides a method for killing cancer cells, which may include: directing a theranostic agent to contact a target cancer cell, imaging the target cancer cell while the theranostic agent is in contact with the target cancer cell, and irradiating the target cancer cell with light while the theranostic agent is in contact with the target cancer cell to kill the target cancer cell. The imaging method may be selected from fluorescence microscopy, bioluminescence imaging, and confocal laser scanning microscopy. The theranostic agent may be combined with a buffer solution before contacting the target cancer cell. The target cancer cell may be present in a living mammal.

[0085] In the step of irradiating the target cancer cells with light, when the theranostic agent contacts the target cancer cells, the theranostic agent may absorb the light and generate active oxygen to kill the target cancer cells.

[0086] Light irradiation may be white light irradiation, red light irradiation, or laser irradiation.

[0087] According to the aforementioned embodiments, the present invention provides a method for halting or inhibiting tumor growth in a mammal. The method may include administering a theranostic agent to the mammal; directing the theranostic agent to contact the tumor site; after contacting the tumor site with the theranostic agent, locating the tumor site using an imaging method; and irradiating the tumor site with light in the presence of the theranostic agent to halt or inhibit tumor growth. The theranostic agent may be administered by injection. The imaging method may include at least one of fluorescence microscopy, bioluminescence imaging, and confocal laser scanning microscopy.

[0088] In the step of irradiating the tumor site with light, when the theranostic agent is present in the tumor site, it can absorb light and generate active oxygen to stop or inhibit the growth of the tumor.

[0089] In the step of irradiating the tumor site with light, when the theranostic agent is present at the tumor site, light energy can be converted into heat energy to stop or inhibit the growth of the tumor.

[0090] Light irradiation may be white light irradiation, red light irradiation, or laser irradiation.

[0091] The following examples are provided to further illustrate and facilitate understanding of the present invention and are not intended to limit the present invention in any way.

[0092] Examples

[0093] Example 1-Synthesis and Characterization

[0094] The synthetic routes of TPA-TBT and TPE-TBT are as follows: Figure 1 shown.

[0095] Synthesis of tributyl(4-(2-butyloctyl)thiophen-2-yl)stannane (Compound 2)

[0096] 3-(2-butyloctyl)thiophene (Compound 1, 2.00 g, 7.93 mmol) was dissolved in anhydrous tetrahydrofuran (80 mL) and then n-butyllithium (n-BuLi) (3.8 mL, 2.5 M in hexane) was added at -78 ° C under a nitrogen atmosphere. The mixture was stirred at the same temperature for one hour, followed by the addition of tributyltin chloride (3.35 g, 10.31 mmol). The reaction was slowly warmed to room temperature and stirred overnight. The reaction mixture was quenched with potassium fluoride (KF) aqueous solution and then washed with water and brine, and dehydrated using sodium sulfate (Na2SO4). The crude product was obtained by concentration under reduced pressure and used without further purification.

[0097] Synthesis of 4-(4-(2-butyloctyl)thiophen-2-yl)-N,N-bis(4-methoxyphenyl)aniline (Compound 3)

[0098] 4-Bromo-N,N-bis(4-methoxyphenyl)aniline (1.00 g, 2.61 mmol), compound 2 (1.70 g, 3.13 mmol), Pd2(dba)3 (120 mg, 0.13 mmol), and P(o-tol)3 (318 mg, 1.04 mmol) were dissolved in anhydrous toluene (30 ml) and heated to 110°C under nitrogen with stirring overnight. After cooling to room temperature, the reaction mixture was extracted with hexane and washed with aqueous potassium fluoride solution, water, and brine. After concentration under reduced pressure, the crude product was purified by column chromatography (stationary phase: silica gel; mobile phase: n-hexane / dichloromethane = 1 / 1) to obtain the product as a light yellow oil (1.19 g, 82%). 1 H NMR (400MHz, CDCl3) δ7.42 (d, J = 8.7Hz, 2H), 7.14–7.05 (m, 4H), 7.02 (s, 1H), 6.98–6.91 (m, 2H), 6.90–6.82 (m, 4H) ),6.77(s,1H),3.83(s,6H),2.55(d,J=6.8Hz,2H),1.70–1.63(m,1H),1.38–1.25(m,16H),0.91(t,J=6.7Hz,6H). 13 C NMR (101MHz, CDCl3) δ155.88,148.00,143.84,142.89,140.84,127.11,126.49,126.33,123.69,120.82,11 9.13,114.71,55.50,38.87,35.18,33.39,33.35,31.94,30.04,29.72,29.66,29.37,26.64,22.71,14.14.

[0099] Synthesis of 4-(4-(2-butyloctyl)-5-(tributyltinyl)thiophen-2-yl)-N,N-bis(4-methoxyphenyl)aniline (Compound 4)

[0100] Compound 3 solution (1.00 g, 1.80 mmol) is placed in anhydrous tetrahydrofuran (20 ml) and n-butyl lithium (0.86 ml, 2.5 M in hexane) is added under nitrogen at -78 ° C. The mixture is stirred for one hour at the same temperature, and then tributyltin chloride (0.76 g, 2.34 mmol) is added. The reaction is slowly warmed to room temperature and stirred overnight. The reaction mixture is quenched with potassium fluoride aqueous solution, then washed with water and brine, and dehydrated using sodium sulfate (Na2SO4). The crude product is obtained by concentration under reduced pressure and can be used without further purification.

[0101] Synthesis of 2-(4-(2,2-bis(4-methoxyphenyl)-1-phenylethene)phenyl)-4-(2-butyloctyl)thiophene (Compound 5)

[0102] A mixture of 4,4'-(2-(4-bromophenyl)-2-phenylethene-1,1-diyl)bis(methoxybenzene) (1.00 g, 2.13 mmol), compound 2 (1.38 g, 2.55 mmol), Pd2(dba)3 (97 mg, 0.11 mmol), and P(o-tol)3 (259 mg, 0.85 mmol) was dissolved in anhydrous toluene (30 mL) and heated to 110°C under nitrogen with stirring overnight. After cooling to room temperature, the reaction mixture was extracted with hexane and washed with aqueous potassium fluoride solution, water, and brine. After concentration under reduced pressure, the crude product was purified by column chromatography (stationary phase: silica gel; mobile phase: n-hexane / dichloromethane = 1 / 1) to obtain the product as a light yellow oil (1.06 g, 77%). 1 H NMR (400MHz, CDCl3) δ7.36 (d, J = 8.4Hz, 2H), 7.15–7.10 (m, 3H), 7.10–7.04 (m, 3H), 7.04–6.99 (m, 4H), 6.99–6.93 (m, 2H), 6.81 (s ,1H),6.71–6.64(m,4H),3.77(d,J=3.9Hz,6H),2.54(d,J=6.8Hz,2H),1.68–1.60(m,1H),1.36–1.24(m,16H),0.94–0.87(m,6H). 13 C NMR (101MHz, CDCl3) δ158.17,158.08,144.18,143.62,143.40,142.97,140.27,138.70,136.36,136.33,132.61,132.22,131.84,131.46,127.7 3,126.16,124.77,124.68,120.11,113.14,112.99,55.09,38.84,35.11 ,33.31,33.01,31.92,29.70,28.87,26.59,23.07,22.69,14.17,14.13.

[0103] Synthesis of 4-(4-(2-butyloctyl)-5-(tributyltinyl)thiophen-2-yl)-N,N-bis(4-methoxyphenyl)aniline (Compound 6).

[0104] A solution of compound 5 (1.00 g, 1.56 mmol) was placed in anhydrous tetrahydrofuran (20 ml) and n-butyl lithium (n-BuLi) (0.75 ml, 2.5 M in hexane) was added under nitrogen at -78 ° C. The mixture was stirred for one hour at the same temperature, and then tributyltin chloride (0.66 g, 2.02 mmol) was added. The reaction was allowed to slowly warm to room temperature and stirred overnight. The reaction mixture was quenched with potassium fluoride (KF) aqueous solution, then washed with water and brine, and dehydrated using sodium sulfate (Na2SO4). The crude product was obtained by concentration under reduced pressure and used without further purification.

[0105] Synthesis of TPA-TBT

[0106] A mixture of 4,8-dibromo-6-(2-ethylhexyl)[1,2,5]thiadiazo[3,4-f]benzotriazole (100 mg, 0.22 mmol), compound 4 (0.76 g, 0.90 mmol), Pd2(dba)3 (10 mg, 0.011 mmol), and P(o-tol)3 (27 mg, 0.090 mmol) was dissolved in anhydrous toluene (10 ml) and stirred at 110°C overnight under nitrogen. After cooling to room temperature, the reaction mixture was extracted with hexane and washed with aqueous potassium fluoride solution, water, and brine. After concentration under reduced pressure, the crude product was purified by column chromatography (stationary phase: silica gel; mobile phase: n-hexane / dichloromethane = 1 / 2) to obtain the product as a dark blue solid (204 mg, 65%). 1 H NMR (400MHz, CDCl3) δ7.52(d,J=8.7Hz,4H),7.25(s,2H),7.17–7.09(m,8H),7.03–6.94(m,4H),6.92–6.84(m,8H),4.81(d,J=7.2Hz,2H),3.8 4(s,12H),2.67(d,J=7.0Hz,4H),2.47–2.38(m,1H),1.55–1.31(m,12H ),1.23–1.12(m,4H),1.10–0.80(m,38H),0.67(td,J=7.0,2.5Hz,6H). 13C NMR (101MHz, CDCl3) δ155.98,151.96,148.28,146.16,144.93,143.73,140.7 3,128.34,126.69,126.53,124.92,120.44,114.74,113.87,61.50,55.51,40 .66,39.10,34.76,33.29,32.92,31.81,30.52,29.55,28.59,28.46,26.41,2 3.81,22.97,22.84,22.65,14.09,14.06,14.04,10.42.MS(ESI)m / zcalcd.for C 86 H 105 N7O4S3 + :1395.7390.Found:1395.7391.

[0107] Synthesis of TPE-TBT

[0108] A mixture of 4,8-dibromo-6-(2-ethylhexyl)[1,2,5]thiadiazo[3,4-f]benzotriazole (100 mg, 0.22 mmol), compound 6 (0.79 g, 0.85 mmol), Pd2(dba)3 (10 mg, 0.011 mmol), and P(o-tol)3 (26 mg, 0.085 mmol) was dissolved in anhydrous toluene (10 ml) and stirred at 110°C overnight under nitrogen. After cooling to room temperature, the reaction mixture was extracted with hexane and washed with aqueous potassium fluoride solution, water, and brine. After concentration under reduced pressure, the crude product was purified by column chromatography (stationary phase: silica gel; mobile phase: n-hexane / dichloromethane = 1 / 2) to obtain the product as a dark blue solid (15 mg, 61%). 1 H NMR (400MHz, CDCl3) δ7.47(d,J=8.4Hz,4H),7.35(s,2H),7.18–6.97(m,22H),6.70(dd,J=15.4,8.8Hz,8H),4.80(d,J=7.2Hz,2H),3.79(d,J=1 3.0Hz,12H),2.66(d,J=7.0Hz,4H),2.45–2.36(m,1H),1.54–1.31(m,12 H),1.23–1.14(m,4H),1.10–0.81(m,38H),0.68(td,J=7.0,2.5Hz,6H). 13C NMR (101MHz, CDCl3) δ158.24,158.13,151.97,145.77,144.94,144.15,143.83,143.79,140.44,1 38.76,136.35,136.31,132.66,132.01,131.85,131.50,129.17,127.77,126.21,125.82,124.96, 113.90,113.18,113.02,61.68,55.10,40.65,39.08,34.70,33.25,32.89,31.81,30.53,29.55,28 .55,28.45,26.37,23.82,22.97,22.84,22.66,14.10,14.06,14.04,10.43.MS(ESI)m / zcalcd.for C 102 H 115 N5O4S3Na + :1593.8037.Found:1593.8024.

[0109] Example 2 - Photophysical, photodynamic and photothermal properties

[0110] First, the absorption and emission spectra were measured. Figure 3a It can be seen that the maximum absorption peaks of TPE-TBT and TPA-TBT are located at 582 and 620 nm, respectively, showing a red shift, which is due to the enhanced donor-acceptor (DA) intensity from TPE-TBT to TPA-TBT. In addition, the maximum emission peaks of the emission spectra of these two molecules are at 750 and 830 nm, respectively (as shown in Figure 2). Figure 3b ). TPA-TBT shows a large Stokes shift of 210 nm, which is beneficial for practical applications. The aggregation behavior of the compounds was then studied. After adding poor solvent water to THF organic solvent, the fluorescence intensity of the two luminescent materials first decreased at low water ratios (0–50% water), which was due to the twisted intramolecular charge transfer (TICT) effect. As the water ratio increased, the fluorescence intensity of the two compounds gradually increased, which was attributed to the typical AIE effect. It is worth noting that TPA-TBT showed more obvious AIE characteristics, with an intensity enhancement of 3 times (such as Figure 3c ).

[0111] In order to better apply it to cancer treatment and diagnosis, TPA-TBT and TPE-TBT were prepared into nanoparticles (NPs) by nanoprecipitation method, and DSPE-PEG2000 was used as the coating matrix. The hydrodynamic diameters of the two nanoparticles were measured by dynamic light scattering (DLS). The results showed that TPA-TBT and TPE-TBT nanoparticles were uniformly dispersed in water, with diameters of approximately 37nm and 45nm, respectively (e.g. Figure 4 and Figure 5 The UV / visible absorption and fluorescence spectra of the two nanoparticles were also measured, and the results were similar to those in THF solution. The fluorescence quantum yields (QY) of the two compounds in solid state, nanoparticles (NPs), tetrahydrofuran (THF) and other solvents were also measured (e.g. Figure 3d ). The results showed that TPA-TBT nanoparticles had a high absolute quantum yield of 7%, while the quantum yield of TPE-TBT nanoparticles was even higher, reaching 28%. The quantum yield of TPA-TBT in the solid state was 13%, which was higher than its quantum yield in nanoparticles and THF solution (5%). In contrast, TPE-TBT had a significant quantum yield in the solution state, indicating that TPE-TBT exhibited a more significant TICT effect and active radioactive decay. To further explore the phototherapeutic potential of these AIEgens, the ROS generation efficiency and photothermal conversion efficiency (PCE) were studied. We utilized a common ROS indicator, dichlorofluorescein diacetate (H2DCF-DA), whose fluorescence can be sensitively activated by ROS to indicate the overall ROS generation. From Figure 3e It can be seen that in the presence of TPA-TBT nanoparticles, the fluorescence intensity of H2DCF-DA increases rapidly. It eventually reaches about 210 times the initial intensity after 5 minutes of white light irradiation, showing a higher ROS generation efficiency than TPE-TBT nanoparticles. In addition, TPA-TBT nanoparticles exhibit better photothermal performance. Figure 3f As shown, after 350 seconds of 660nm laser irradiation, TPA-TBT nanoparticles reached a temperature of approximately 57°C, with a photothermal conversion efficiency (PCE) of 44%. In contrast, the PCE of TPE-TBT nanoparticles was only 30%. Taking all factors into consideration, TPA-TBT nanoparticles exhibit longer fluorescence wavelengths, higher reactive oxygen species generation efficiency, and better photothermal efficiency. Therefore, TPA-TBT nanoparticles hold great promise for application in optical diagnostics and therapeutics for synergistic cancer treatment.

[0112] Example 3 - Cell Imaging

[0113] The cytotoxicity of TPA-TBT nanoparticles in a dark environment was investigated using CCK-8 assay to explore the biocompatibility of the synthesized probe. Figure 6a As shown in the results, even at very high concentrations (100 μg / mL), TPA-TBT nanoparticles showed no obvious dark toxicity to normal cells (human embryonic kidney cells 293T) and tumor cells (human cervical cancer cells Hela and mouse breast cancer cells 4T1). However, after additional treatment with weak white light irradiation, TPA-TBT nanoparticles showed obvious phototoxicity to cells (e.g. Figure 6b ). Even at low concentrations of nanoparticles, cell viability was still significantly reduced. These observations indicate that TPA-TBT nanoparticles have excellent biocompatibility and effective photokilling effects. Flow cytometry and confocal imaging further confirmed its photokilling efficiency against cancer cells, showing that TPA-TBT nanoparticles combined with photothermal therapy (PTT) and photodynamic therapy (PDT) were effective, with a total killing rate of up to 67.3%. 4T1 cells from other experimental groups (including untreated, light-treated only, and nanoparticle-treated only) showed almost no significant apoptotic or necrotic fractions in the overall cell population (such as Figure 6c ).

[0114] In addition, the generation of intracellular reactive oxygen species (ROS) was investigated by using H2DCF-DA in 4T1 cells and irradiating them with light. Figure 7 As shown, 4T1 cells treated with TPA-TBT nanoparticles and H2DCF-DA exhibited strong green fluorescence under weak white light irradiation. In contrast, no fluorescence was detected in cells treated with PBS buffer (control group) or in cells treated with TPA-TBT nanoparticles alone without laser irradiation. These results indicate that TPA-TBT nanoparticles can effectively generate reactive oxygen species, thereby exhibiting excellent phototoxicity against cancer cells.

[0115] Example 4 - Image-guided in vivo cancer therapy

[0116] To investigate the in vivo fluorescence imaging (FLI) and tumor accumulation ability of TPA-TBT nanoparticles, the nanoparticles were first injected intravenously into 4T1 tumor-bearing mice. Fluorescence images of the mice were acquired at different time intervals after injection. Figure 8a and 8bAs shown in the figure, the fluorescence signal gradually increased with time and reached the strongest point 12 hours after injection, indicating that TPA-TBT nanoparticles can accumulate in the tumor site and show excellent performance in FLI. In order to further study the biodistribution of TPA-TBT nanoparticles, the main organs (heart, liver, spleen, lung and kidney) and tumor tissues of mice were collected for analysis 12 hours after the injection of nanoparticles. The results showed that TPA-TBT nanoparticles mainly accumulated in tumors and liver. Among them, the tumor tissue showed the strongest fluorescence signal, indicating the tumor accumulation ability and liver metabolic effect of TPA-TBT nanoparticles (such as Figure 9 ).

[0117] The in vivo optical diagnostic and therapeutic performance and anti-tumor ability of TPA-TBT nanoparticles in 4T1 tumor mice were then studied. First, the tumor temperature of mice treated with TPA-TBT nanoparticles rose rapidly from 35°C to above 50°C within 2 minutes after laser irradiation, and reached about 57°C within 10 minutes of irradiation. In contrast, the tumor temperature of the control group (treated with PBS buffer only) did not change significantly (such as Figure 8c 、 8d and Figure 10 To investigate the sustained therapeutic effect of TPA-TBT nanoparticles, mice bearing 4T1 tumors were randomly divided into four groups for different treatments. Figure 8e and Figure 11 As shown in Figure 2, tumor images were taken before (day 0) and after (days 1, 7, and 14) injection of TPA-TBT nanoparticles. Tumor size was monitored every two days to evaluate the therapeutic effect of each group. 2 ) irradiated group, the tumors were completely eliminated, while the tumors in the other three groups continued to grow with no obvious inhibitory effect. In addition, the body weight of mice in all groups did not show a significant decrease within 14 days, indicating that the TPA-TBT nanoparticle treatment method has good biosafety (such as Figure 12 ).

[0118] In addition, to investigate the tumor killing mechanism during phototherapy, hematoxylin-eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining were performed (e.g. Figure 8fCompared with the three control groups, the tumor cell density in the group receiving TPA-TBT nanoparticles and laser treatment was significantly reduced. Furthermore, TUNEL staining results further confirmed that TPA-TBT nanoparticles combined with laser irradiation could generate reactive oxygen species (ROS) and induce tumor cell apoptosis. These results suggest that TPA-TBT nanoparticles achieve tumor elimination through cell necrosis and apoptosis, and that these effects are due to the simultaneous administration of photothermal therapy (PTT) and photodynamic therapy (PDT) under light conditions.

[0119] In addition, a systematic evaluation of the in vivo safety of the nanoparticles was conducted. Mice were injected with TPA-TBT nanoparticles (10 mg / kg, 200 μL). Blood routine and biochemical analyses were performed on days 7 and 14 after injection and compared with the control group treated with PBS (eg Figure 13 and 14 ). From the analysis, it can be seen that all parameters were within the normal range compared with the control group. In addition, the main organ tissues were collected at different time points for H&E staining, and no abnormalities were found after TPA-TBT nanoparticle treatment (such as Figure 15 These results indicate that TPA-TBT nanoparticles exhibit excellent in vivo biocompatibility, which enables them to be used as highly efficient fluorescence imaging-guided photothermal therapy (PTT) and photodynamic therapy (PDT) agents for widespread application in cancer optical diagnosis and treatment.

[0120] The above embodiments are only used to illustrate the principles of the present invention and should not be understood as any limitation to the present invention. The above embodiments can be modified by a person skilled in the art without departing from the scope of the present invention as defined by the following claims.

Claims

1. A fluorescent compound having aggregation-induced emission properties, wherein the fluorescent compound has the following main structural formula: wherein each of R1, R2 and R3 is independently selected from the following: linear, branched, cyclic alkyl, alkylphenyl, alkylthienyl and other alkyl aromatic groups having 2 to 40 carbon atoms, and wherein one or more non-adjacent carbon atoms are optionally replaced by –O–, –S–, –C(O)–, –C(O–)–O–, –O–C(O)–, –O–C(O)–O– or –C≡C–, and wherein one or more hydrogen atoms are optionally replaced by fluorine (F), chlorine (Cl), bromine (Br), iodine (I) or cyanide (CN), or represents an aromatic, heteroaromatic, aryloxy, heteroaryloxy, aromatic carbonyl, heteroaromatic carbonyl, aromatic carbonyloxy, heteroaromatic carbonyloxy, aryloxycarbonyl or heteroaryloxycarbonyl group having a ring of 4 to 30 atoms, and these ring molecules are unsubstituted or substituted by one or more non-aromatic groups; and Wherein each of R4 and R5 is independently selected from the following combinations:

2. The fluorescent compound according to claim 1, wherein The R1, R2 and R3 are branched alkyl groups having 2 to 40 carbon atoms.

3. The fluorescent compound according to claim 1, wherein The fluorescent compound is selected from one of the following groups:

4. The fluorescent compound according to claim 3, wherein The fluorescent compound exhibits near-infrared luminescence.

5. The fluorescent compound according to claim 1, wherein The fluorescent compound exists in the form of nanoparticles.

6. A therapeutic diagnostic agent comprising the fluorescent compound of claim 1 in nanoparticle form and a polymer matrix encapsulating the fluorescent compound.

7. A method of killing cancer cells, comprising: directing the therapeutic diagnostic agent according to claim 6 to contact the target cancer cells; imaging the target cancer cells when the theranostic agent contacts the target cancer cells using an imaging method selected from the group consisting of: fluorescence microscopy, bioluminescence imaging, and confocal laser scanning microscopy; and While the theranostic agent is in contact with the target cancer cells, the target cancer cells are irradiated with light to kill the target cancer cells.

8. The method according to claim 7, wherein While the theranostic agent is in contact with the target cancer cells, light is irradiated on the target cancer cells, and the theranostic agent absorbs the light and generates reactive oxygen species to kill the target cancer cells.

9. The method according to claim 1, wherein While the theranostic agent is in contact with the target cancer cells, light is irradiated to the target cancer cells, and the theranostic agent converts the light into heat to kill the target cancer cells.

10. The method according to claim 1, wherein The target cancer cell is present in a living mammal.

11. The method according to claim 1, wherein The light irradiation is white light irradiation.

12. The method according to claim 1, wherein The light irradiation is red light irradiation.

13. The method according to claim 12, wherein: The light irradiation is laser irradiation.

14. A method for stopping or inhibiting tumor growth in a mammal, comprising: administering the therapeutic diagnostic agent according to claim 6 to a mammal; directing the theranostic agent to contact the tumor site; After contacting the tumor site with the theranostic agent, locating the tumor site using an imaging method; and In the presence of the theranostic agent at the tumor site, light is irradiated at the tumor site to stop or inhibit tumor growth.

15. The method according to claim 14, wherein When the therapeutic diagnostic agent is present in a tumor site, the tumor site is irradiated with light, and the therapeutic diagnostic agent absorbs the light and generates reactive oxygen species, thereby stopping or inhibiting the growth of the tumor.

16. The method according to claim 1, wherein In the presence of the theranostic agent at the tumor site, light is irradiated to the tumor site, and the theranostic agent converts the light into heat to stop or inhibit the growth of the tumor.

17. The method according to claim 16, wherein The theranostic agent is administered by injection.

18. The method according to claim 17, wherein The imaging method includes at least one of fluorescence microscopy, bioluminescence imaging, and confocal laser scanning microscopy.

19. The method according to claim 18, wherein The light irradiation is white light irradiation.

20. The method according to claim 19, wherein The light irradiation is red light irradiation.

21. The method according to claim 19, wherein The light irradiation is laser irradiation.

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