Amphiphilic AIE light diagnosis and treatment agent as well as preparation method and application thereof

Abstract

The invention discloses an amphiphilic AIE light diagnosis and treatment agent as well as a preparation method and application thereof. The amphiphilic AIE light diagnosis and treatment agent comprises a compound with a structural formula as shown in a formula I, or a pharmaceutically acceptable salt, a solvate and a tautomer thereof, wherein R is n # imgabs0 # connected groups, n is greater than or equal to 1 and less than or equal to 5, and wavy lines represent joints; x is selected from one or more of O, S and Se. According to the AIE light diagnosis and treatment agent, active oxygen generated through light induction is combined with the photo-thermal effect to cooperatively kill tumor cells, the effect of inhibiting tumor growth is achieved, and I-type photodynamic therapy independent of oxygen is combined with photo-thermal therapy mainly (the photo-thermal temperature can be rapidly increased to 60 DEG C under the low illumination intensity (300 mW / cm < 2 >); meanwhile, good near-infrared I / II region fluorescence imaging, photoacoustic imaging and photo-thermal imaging can be achieved, and the compound is a light diagnosis and treatment agent with unique hypoxia resistance and can effectively achieve tumor treatment in a short time.

Description

Technical Field

[0001] The present invention relates to the field of medical technologies, and particularly to an amphiphilic AIE photodiagnostic agent with the synergistic effect of photothermal and type I reactive oxygen species, a preparation method thereof, and an application thereof. Background Art

[0002] As a new type of disease diagnosis and treatment technology, photodiagnosis and treatment plays an important role in real-time biomedical imaging diagnosis and monitoring of tumors and in situ precise treatment. Among them, the biomedical imaging diagnosis methods mainly include fluorescence imaging (FLI), photoacoustic imaging (PAI), and photothermal imaging (PTI), which have the advantages of non-invasiveness, high sensitivity, fast and efficient, low cost, real-time monitoring, spatial resolution, high temporal resolution, and tissue penetration, and have been widely used in biomedical and clinical medicine fields such as subcellular localization analysis and fluorescence surgical navigation. As a new type of minimally invasive treatment method, phototherapy is mainly divided into two types: photodynamic therapy (PDT) and photothermal therapy (PTT). Photodynamic therapy is mainly that a photodiagnostic agent generates singlet oxygen ( 1 O2) through energy conversion or forms free radicals (·OH, ·O2ˉ, H2O2, etc.) through electron transfer to destroy biological macromolecules, which are respectively called type I PDT and type II PDT. That is, type I PDT can generate hydroxyl radicals and superoxide radicals and does not require oxygen dependence, while type II PDT not only requires a photodiagnostic agent and an energy source, but also mainly depends on sufficient oxygen. However, the microenvironment of malignant tumors is a rare hypoxic characteristic, which limits the effect of PDT on cancer. Type I PDT has become the preferred method for phototherapy of tumors because it is not limited by oxygen. Photothermal therapy (PTT) can effectively accelerate blood circulation in the irradiated tumor site and further improve the problem of tumor hypoxia and oxygen consumption in PDT. Therefore, the PDT-PTT technology formed by combining PDT and PTT can greatly improve the phototherapy effect. Due to its unique optical properties, reactive oxygen species yield, and photothermal conversion efficiency and other advantages, aggregation-induced emission (AIE) materials have been widely used in the field of biomedicine. Especially, AIE molecules as photodiagnostic agents have received extensive attention and development in the real-time diagnosis, monitoring, and treatment of malignant tumors, but the development of high-performance multifunctional AIE photodiagnostic systems is still very limited, and there are huge challenges in their design and development. This is mainly because a multifunctional AIE system with excellent performance should be able to simultaneously achieve FLI, PAI, PTT, and PDT. According to the Jablonski diagram, there is competition between their radiative and non-radiative decay processes. Therefore, how to tune the balance between radiative and non-radiative decay processes and synchronously achieve FLI, PAI, PTT, and PDT is one of the key factors in the design and development of multifunctional photodiagnostic agents. Summary of the Invention

[0003] Object of the Invention

[0004] To overcome the above deficiencies, the object of the present invention is to provide an amphiphilic AIE phototheranostic agent with the synergistic effect of photothermal and type I reactive oxygen species, and its preparation method and application.

[0005] The preparation method of the AIE phototheranostic agent of the present invention is simple and easy to synthesize; in vitro, the AIE phototheranostic agent of the present invention not only exhibits good ability to specifically target lysosomes of breast cancer cells (MDA-MB-231), but also has a significant inhibitory effect on tumors; in vivo, the AIE phototheranostic agent of the present invention shows excellent fluorescence imaging, photoacoustic imaging and photothermal imaging capabilities for malignant tumors; the AIE phototheranostic agent of the present invention ablates and inhibits tumors by rapidly generating ROS through light induction combined with photothermal effect to achieve the therapeutic purpose of inhibiting tumor growth, and is mainly based on type I photodynamic therapy combined with photothermal therapy that generates reactive oxygen species independent of oxygen (the photothermal temperature can be rapidly increased to 60 °C under a lower light intensity (635 nm laser, 300 mW / cm 2 ). Therefore, the AIE phototheranostic agent of the present invention is a phototheranostic agent with excellent hypoxia tolerance, and can effectively achieve the comprehensive treatment of malignant tumors for diagnostic monitoring and optical imaging-guided type I photodynamic therapy combined with photothermal therapy in a short time, thereby greatly improving the treatment efficiency of malignant tumors.

[0006] Solution

[0007] To achieve the object of the present invention, the technical solution adopted by the present invention is as follows:

[0008] In the first aspect, the present invention provides an amphiphilic AIE phototheranostic agent with the synergistic effect of photothermal and type I reactive oxygen species, which includes a compound with a structural formula as shown in Formula I, or a pharmaceutically acceptable salt thereof, a solvate thereof and a tautomer thereof;

[0009]

[0010] Among them, R is: n connected groups, where 1 ≤ n ≤ 5, and the wavy line represents the connection point;

[0011] X is selected from one or more of O, S and Se.

[0012] Further optionally: 1 ≤ n ≤ 3, optionally n = 2 or 3.

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

[0014]

[0015]

[0016] Furthermore, it is a nano-diagnostic and therapeutic agent, optionally prepared from the compound shown in Formula I by self-assembly technology. In the second aspect, a method for preparing the amphiphilic AIE photo-diagnostic and therapeutic agent described in the first aspect is provided, including the following steps: In a solvent, under the action of a basic substance, the compounds shown in Formula A and Formula B generate the compound shown in Formula I:

[0017]

[0018] Furthermore, the solvent is selected from one or more of N,N-dimethylformamide, dimethyl sulfoxide, toluene, o-xylene, dichloromethane, and chloroform; optionally, the solvent is a super-dry solvent.

[0019] And / or, the basic substance is selected from one or more of pyridine, triethylamine, potassium carbonate, and sodium carbonate;

[0020] And / or, the reaction is carried out in an anaerobic atmosphere;

[0021] And / or, the reaction temperature is 100-200 °C, optionally 120-180 °C, optionally 140-160 °C, optionally 150-160 °C;

[0022] And / or, the reaction time is 10-18 h, optionally 12-18 h;

[0023] And / or, the molar ratio of the compounds shown in Formula A and Formula B is 1:0.9-1.5, optionally 1:1-1.5, optionally 1:1-1.2.

[0024] Furthermore, the compound shown in Formula I is prepared into nanoparticles: The compound shown in Formula I is dissolved in an organic solvent and deionized water, ultrasonicated, dialyzed, and filtered to obtain nanoparticles;

[0025] Optionally, the organic solvent is selected from one or more obtained from DMSO, dimethyl sulfoxide, and tetrahydrofuran;

[0026] Optionally, the ultrasonic time is 2-40 min, optionally 30-40 min;

[0027] Optionally, the ultrasonic power is 20-300 W.

[0028] Optionally, dialysis is carried out in deionized water, optionally, the deionized water is changed once every 6-10 h, and optionally, dialysis is carried out for 24-56 h;

[0029] Optionally, the pore size of the filtration is 0.1-0.8 μM, optionally 0.22 μM.

[0030] Further, the preparation method of the compound shown in formula B includes: reacting 1,3 - propane sultone and 2 - methylbenzothiazole in a solvent to obtain the compound shown in formula B;

[0031] Optionally, in the preparation of the compound shown in formula B, the solvent is toluene;

[0032] Optionally, in the preparation of the compound shown in formula B, the reaction is carried out in an inert gas atmosphere;

[0033] Optionally, the reaction temperature is 45 - 100 °C and the reaction time is 6 - 10 h;

[0034] Optionally, the molar ratio of 1,3 - propane sultone to 2 - methylbenzothiazole is 1:0.9 - 1.5, optionally 1:1 - 1.5, optionally 1:1 - 1.2.

[0035] In the third aspect, there is provided an application of the amphiphilic AIE photodiagnostic agent described in the first aspect, or the amphiphilic AIE photodiagnostic agent prepared by the preparation method described in the second aspect, in the preparation of an aggregation - induced emission - type photodiagnostic agent, or a product targeting tumor cell lysosomes, or an anti - tumor product combining type I photodynamic therapy and photothermal therapy, or a tumor fluorescence - photothermal imaging - guided photothermal - photodynamic synergistic therapy product, or a photodiagnostic agent product mainly based on type I photodynamic therapy for tumors.

[0036] In the fourth aspect, there is provided an application of the amphiphilic AIE photodiagnostic agent described in the first aspect, or the amphiphilic AIE photodiagnostic agent prepared by the preparation method described in the second aspect, in the preparation of a photodynamic - photothermal combined anti - tumor photodiagnostic agent mediated by near - infrared I / II region fluorescence imaging, photoacoustic imaging, and photothermal imaging, or an anti - tumor product for photothermal therapy, or a drug for photothermal - type I photodynamic synergistic ablation therapy of malignant tumors; optionally, the photothermal temperature of the amphiphilic AIE photodiagnostic agent under a light intensity of 300 mW / cm 2 can reach 56 - 61 °C, optionally 59 - 61 °C. Optionally, the use concentration of the amphiphilic AIE photodiagnostic agent is 0.1 - 20 μM, optionally 1 - 16 μM, optionally 0.5 - 2 μM, optionally 1 - 3 μM.

[0037] Beneficial Effects

[0038] (1) The amphiphilic AIE photo-theranostic agent (nanoparticle) of the present invention has unique aggregation-induced emission characteristics, amphiphilicity and excellent photostability; it has significant ROS generation ability in vitro (and mainly generates reactive oxygen species mainly for type I PDT treatment (such as ·O2ˉ, ·OH), and type I PDT is not limited by oxygen supply) and efficient photothermal conversion ability (it can rapidly heat up to 60 °C in a short time); and it has good biocompatibility and low cytotoxicity; it shows good targeting of tumor cell lysosomes and significant photodynamic therapy ability under in vitro conditions; it can show excellent fluorescence imaging and photothermal imaging abilities at the in vivo level; it kills tumor cells by rapidly generating ROS through electron transfer combined with photothermal effects, achieving the treatment purpose of inhibiting tumor growth, and mainly relies on type I photodynamic therapy (PDT) without oxygen combined with photothermal therapy, which is a photo-theranostic agent with good hypoxia tolerance characteristics. It can generate reactive oxygen species mainly for type I PDT treatment (such as ·O2ˉ, ·OH) (type I PDT is not limited by oxygen supply) through light induction, and has a high photothermal conversion efficiency, which can effectively rapidly heat the local tumor to 60 °C in a short time, achieving the purpose of killing tumors and thus improving the treatment efficiency. Most of the existing phototherapeutic agents mainly rely on type II photodynamic therapy that requires oxygen to form reactive oxygen species (ROS), resulting in poor treatment effects due to tumor hypoxia.

[0039] (2) The amphiphilic AIE photo-theranostic agent (nanoparticle) of the present invention is an aggregation-induced emission type photo-theranostic agent with type I reactive oxygen species and photothermal generation ability. The temperature of its photothermal treatment can reach 56 - 61 °C, optionally up to 59 - 6I °C. It is a novel amphiphilic aggregation-induced emission (AIE) nano-theranostic agent with both type I reactive oxygen species and photothermal conversion ability. This theranostic agent has excellent fluorescence imaging, photoacoustic imaging and photothermal imaging characteristics, and at the same time has the effects of photothermal therapy (PTT) and mainly type I photodynamic therapy (PDT), and is thus used for the integrated diagnosis and treatment of tumors. The AIE photo-theranostic agent of the present invention can balance the energy consumption of radiative and non-radiative conversion, can greatly improve the comprehensive curative effect of the photo-theranostic agent on hypoxic solid tumors, and provides a new strategy for the development of novel multifunctional phototherapy systems.

[0040] (3) The preparation method of the photo-theranostic agent of the present invention uses easily available raw materials, has mild synthesis conditions, a simple preparation method and convenient purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] One or more embodiments are illustrated by the pictures in the corresponding drawings. These illustrative descriptions do not constitute a limitation on the embodiments. The special word "exemplary" here means "serving as an example, an embodiment or an illustration". Any embodiment described as "exemplary" here does not have to be construed as superior to or better than other embodiments.

[0042] Figure 1 It is the 1H NMR spectrum of the compound TTCMSB prepared in Example 1 of the present invention.

[0043] Figure 2 It is the high-resolution mass spectrum of the compound TTCMSB prepared in Example 1 of the present invention.

[0044] Figure 3 It is the 1H NMR spectrum of the compound TPTMSB prepared in Example 2 of the present invention.

[0045] Figure 4 It is the high-resolution mass spectrum of the compound TPTMSB prepared in Example 2 of the present invention.

[0046] Figure 5 It is the 1H NMR spectrum of the compound TPAMSB prepared in Example 3 of the present invention.

[0047] Figure 6 It is the high-resolution mass spectrum of the compound TPAMSB prepared in Example 3 of the present invention.

[0048] Figure 7 It is the fluorescence excitation spectrum and emission spectrum of the compounds TTCMSB and TPTMSB prepared in Test Example 1 of the present invention. Among them, (a) is the fluorescence excitation spectrum of TPTMSB, (b) is the emission spectrum of TPTMSB; (c) is the fluorescence excitation spectrum of TTCMSB, (d) is the emission spectrum of TTCMSB.

[0049] Figure 8 It is the AIE performance curve of the compound TTCMSB prepared in Test Example 1 of the present invention, where f H is the proportion of n-hexane in the mixed solution.

[0050] Figure 9 It is the experimental graph of the cytotoxicity and photodynamic therapy of different concentrations of compound TTCMSB NPs nanoparticles on MDA-MB-231 cells in Test Example 2 of the present invention. Among them, the black bar graph (Dark) represents the cytotoxicity under dark conditions (left); the purple bar graph (Light) represents the photodynamic therapy effect (right).

[0051] Figure 10 It is the experimental graph of the photodynamic therapy of different concentrations of TTCMSB NPs nanoparticles on MDA-MB-231 cells measured by flow cytometry in Test Example 2 of the present invention.

[0052] Figure 11 It is the in vitro ROS generation result graph of compound TTCMSB NPs nanoparticles in Test Example 3 of the present invention. Among them, (a) is the singlet oxygen generated by TTCMSB NPs ( 1ESR signals of O2); (b) is the ESR signal of hydroxyl radical (·OH) generated by TTCMSB NPs, (c) is the ESR signal of superoxide radical (·O2ˉ) generated by TTCMSB NPs, (d) is the total ROS amount measured by DCFH probe; (e) is the amount of hydroxyl radical in the system measured by HPF probe, (f) is the amount of superoxide radical in the system measured by DHR probe.

[0053] Figure 12 are the fluorescence imaging and photothermal imaging diagrams of TTCMSB NPs nanoparticles in MDA-MB-231 tumor-bearing mice in vivo at different times in Test Example 4 of the present invention; where a is the fluorescence imaging of tumor-bearing mice in vivo, and b is the photothermal imaging diagram of tumor-bearing mice in vivo.

[0054] Figure 13 is the treatment effect diagram of TTCMSB NPs nanoparticles in MDA-MB-231 tumor-bearing mice in Test Example 4 of the present invention. a is the change diagram of mouse tumor volume, and b is the change diagram of mouse body weight.

[0055] Figure 14 is the photothermal imaging diagram of nanoparticles prepared from TPTMSB and TTCMSB in MDA-MB-231 tumor-bearing mice in vivo in Test Example 4 of the present invention. Detailed implementation manners

[0056] 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. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0057] In addition, for better illustration of the present invention, numerous specific details are given in the following detailed implementation manners. Those skilled in the art should understand that the present invention can be implemented without some specific details. In some embodiments, the raw materials, schemes, methods, means, etc. well-known to those skilled in the art are not described in detail to highlight the gist of the present invention.

[0058] Unless otherwise clearly stated, throughout the specification and claims, the term "comprise" or its variations such as "comprises" or "including" etc. will be understood to include the stated elements or components, without excluding other elements or other components.

[0059] In the following embodiments, "ultra-dry" in the ultra-dry solvent refers to a solvent that is anhydrous and anaerobic, and has been dried and deoxygenated.

[0060] Example 1: TTCMSB

[0061] 1. Preparation of Compound TTCMSB:

[0062] The general structural formula of the compounds provided by the present invention is:

[0063]

[0064] The preparation method of such novel amphiphilic aggregation-induced emission materials based on thiophene-based electron acceptor derivatives, taking those with two thiophene groups ( ) as an example, includes the following steps:

[0065] (1) Put 0.61 g of 1,3-propane sultone and 0.73 g of 2-methylbenzothiazole into a 250 mL round-bottom flask, add 80 mL of ultradry toluene solution to dissolve, use nitrogen as the protective gas, and react at 100 °C for 8 h. Cool and collect the precipitate, wash it with tetrahydrofuran 2-3 times, and then dry it to obtain the intermediate MSB as described in the following formula;

[0066]

[0067] (2) Dissolve 0.355 g of triphenylamine-2,2'-bithiophene-5'-aldehyde intermediate (TTC) and 0.271 g of intermediate MSB in 50 mL of ultradry N,N-dimethylformamide (DMF), use pyridine as the catalyst, and react at 150 °C for 12 h in a nitrogen environment to obtain the compound shown as TTCMSB;

[0068]

[0069] (3) Purify it by silica gel column to obtain a solid sample;

[0070] 2. Preparation of TTCMSB NPs:

[0071] (1) First, weigh 5.0 mg of the sample shown as TTCMSB (or formula III-1), dissolve it in 2 mL of DMSO, and then add 8 mL of deionized water and ultrasonicate it in an ultrasonic instrument for 30 min;

[0072] (2) After completely removing the undissolved particles, put it into a dialysis bag and dialyze it in deionized water, change the sterile water dialysis solution every 8 h, and dialyze for a total of 48 h. After 2 days, filter the aqueous solution of nanoparticles with a syringe equipped with a filter (0.2 μM);

[0073] 3. Characterization of the solid product obtained in Example 1, namely TTCMSB (or Compound of Formula III-1) , and the 1H NMR results are as shown in Figure 1 , which determined the chemical structure of TTCMSB; the specific NMR data are as follows: 11H NMR (400 MHz, DMSO-D6) δ 8.46 - 8.31 (m, 3H), 8.03 (d, J = 4.1 Hz, 1H), 7.91 - 7.80 (m, 2H), 7.74 (t, J = 7.7 Hz, 1H), 7.65 - 7.57 (m, 4H), 7.50 (d, J = 4.0 Hz, 1H), 7.35 (t, J = 7.9 Hz, 4H), 7.09 (m, 6H), 7.00 - 6.95 (m, 2H), 5.03 (t, J = 7.8 Hz, 2H), 2.65 (t, J = 6.6 Hz, 2H), 2.18 (m, 2H).

[0074] The sample solution was added dropwise to dryness on a mass spectrometer sample plate, and analyzed using a high performance liquid chromatography - electrospray - ion trap / time of flight tandem mass spectrometer. The results were as Figure 2 , and the results showed that: the theoretical value of the molecular weight of TTCMSB was 690.92, then (M + H)[m / z]: was 691.1173, and the actual value was 691.11, with the theoretical value being consistent with the actual value.

[0075] Example 2: TPTMSB

[0076] The difference from Example 1 was that the intermediate TTC was replaced with the intermediate TPT (i.e., R was 1 ), and other steps were referred to Example 1 to obtain the TPTMSB compound of the following formula, and TPTMSB NPs nanoparticles were prepared.

[0077]

[0078] The product of the TPTMSB compound of the above formula in this Example 2 was characterized, and the 1H NMR results were as Figure 3 , and the chemical structure of TPTMSB was determined. The specific NMR data was as follows:

[0079] 1 1H NMR (400 MHz, CDCl3) δ 9.84 (s, 1H), 7.70 (d, J = 4.0 Hz, 1H), 7.51 (d, J = 8.6 Hz, 3H), 7.35 – 7.23 (m, 10H), 7.16 – 7.11 (m, 7H), 7.11 – 7.02 (m, 8H), 1.59 (s, 1H), 1.39 (d, J = 6.3 Hz, 1H).

[0080] TPTMSB was dissolved in an ethanol solution and diluted to a concentration of 10 μM. The sample solution was added dropwise to dryness on a mass spectrometer sample plate, and analyzed using a high performance liquid chromatography - electrospray - ion trap / time of flight tandem mass spectrometer. The specific results were as attached Figure 4As shown: The theoretical value of the molecular weight of TPTMSB is 608.13, then (M+H)[m / z]: is 631.1311, and the actual value is 631.1328, and the theoretical value is consistent with the actual value.

[0081] Example 3: TPAMSB

[0082] The difference from Example 1 is that the intermediate TTC is replaced with the intermediate TPA (i.e., R is 0 ), and other steps refer to Example 1, to obtain the TPAMSB compound of the following formula, and prepare TPAMSB NPs nanoparticles.

[0083]

[0084] Characterize the solid product of TPAMSB obtained in the above formula of this Example 3, and the hydrogen spectrum NMR results are as Figure 5 shown, and the chemical structure of TPAMSB is determined; the specific NMR data are as follows:

[0085] 1 H NMR(400MHz,DMSO-d6)δ8.36(d,J=8.6Hz,1H),8.31(d,J=8.5Hz,1H),8.17–7.93(m,4H),7.85–7.79(m,1H),7.73(t,J=7.7Hz,1H),7.48–7.40(m,4H),7.27–7.19(m,6H),6.87(d,J=8.7Hz,2H),5.02(t,J=7.9Hz,2H),2.68–2.61(m,2H),2.23–2.11(m,2H).

[0086] Drop the sample solution on the mass spectrometer sample plate until dry, and analyze it using a high-performance liquid chromatography - electrospray - ion trap / time-of-flight tandem mass spectrometer; the specific results are as attached Figure 6 shown: The theoretical value of the molecular weight of TPAMSB is 526.67, then (M+H)[m / z]: is 527.1435, and the actual value is 526.14, and the theoretical value is consistent with the actual value.

[0087] Test Example 1

[0088] 1) Use a fluorescence spectrometer to detect the fluorescence excitation spectrum and emission spectrum of TTCMSB and TPTMSB respectively, and the results are as Figure 7 .

[0089] Figure 7 (a) The results show that the TPTMSB molecule exhibits a characteristic absorption peak at 585 nm, Figure 7(b) The results show that the optimal position of the fluorescence emission peak of the TPTMSB molecule is at 690 nm, accompanied by a Stokes shift of 105 nm. This indicates that the TPTMSB molecule has significant strong ultraviolet absorption and far red / near infrared emission characteristics.

[0090] Figure 7 (c) The results show that the optimal absorption peak of the TTCMSB molecule is at about 550 nm. Figure 7 (d) The results show that the fluorescence emission peak of the TTCMSB molecule is at 850 nm, resulting in a red shift of nearly 300 nm, and the emission band extends to 1100 nm, covering the near infrared I and II regions. This makes the TTCMSB molecule have potential application value in fluorescence imaging in the near infrared I and II regions.

[0091] It shows that different numbers of thiophene groups and sulfonic acid groups have different effects on the optical properties of the molecule, resulting in different emission wavelengths of the molecule. Among them, the emission wavelength of the TTCMSB molecule is longer, reaching 870 nm, that is, fluorescence imaging in the near infrared I region can be achieved, and fluorescence imaging in the near infrared II region can also be achieved, effectively increasing the tissue penetration depth, and at the same time, the effect of photoacoustic imaging can be achieved.

[0092] 2) Measure the fluorescence intensity distribution in solvents with different ratios of dichloromethane / n-hexane:

[0093] Dissolve TTCMSB in a mixed solution of a good solvent / bad solvent. Dichloromethane is the good solvent and n-hexane is the bad solvent. Measure its fluorescence intensity distribution in mixed solutions of dichloromethane / n-hexane with different ratios (f H = 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90%, f H is the proportion of n-hexane in the mixed solution), and detect the change in the fluorescence emission intensity of the TTCMSB molecule under different ratios of dichloromethane / n-hexane through a fluorescence spectrometer. The specific results are as Figure 8 shown. Figure 8 The results show that the TTCMSB molecule has weak fluorescence emission in pure dichloromethane solution. With the gradual addition of the bad solvent n-hexane, the fluorescence emission gradually increases, indicating that the TTCMSB molecule has the characteristics of aggregation-induced emission and excellent AIE performance.

[0094] Test Example 2

[0095] Detect the cytotoxicity test of TTCMSB NPs on MDA-MB-231 cells and the photodynamic therapy effect on the MDA-MB-231 cell line by the CCK-8 method. (MDA-MB-231 cells were purchased from Beina Biotechnology Co., Ltd.). The specific experimental steps are as follows:

[0096] Toxicity Test of TTCMSB NPs on MDA-MB-231 Cells

[0097] (1) At a cell density of 5×10 3 cells per well, inoculate the MDA-MB-231 cell line into a 96-well plate.

[0098] (2) After culturing for 24 h under dark conditions, discard the medium and add fresh DMEM medium containing different concentrations of TTCMSB NPs (0, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM).

[0099] (3) Continue to culture for 24 h, then remove the medium and wash twice with PBS. Add 10 μL of CCK-8 solution and 90 μL of colorless DMEM medium to each well. After incubating for 45 min, measure the absorbance at a wavelength of 450 nm using a microplate reader.

[0100] (4) To further evaluate the effect of TTCMSB NPs on cell viability, flow cytometry (FCM) was used to quantitatively analyze the survival rate and apoptosis rate of MDA-MB-231 cells after incubation with different concentrations of TTCMSB NPs, as shown by the black columns in Figure 9 .

[0101] Photodynamic Therapy Effect of TTCMSB NPs on MDA-MB-231 Cell Line

[0102] (1) In the in vitro photodynamic therapy experiment, inoculate MDA-MB-231 cells into a 96-well plate at a density of 5×10 3 cells per well and culture in an incubator for 24 h.

[0103] (2) Subsequently, remove the medium and add fresh DMEM medium containing different concentrations of TTCMSB NPs (0, 1 μM, 2 μM, 4 μM, 8 μM, and 16 μM) as the therapeutic agent. After incubating for 8 h, remove the medium and treat the cells with different light intensities. The light cycle is once every 2 min, and the total light exposure time is 10 min.

[0104] (3) After treatment, replace with fresh DMEM medium and continue to incubate the cells for 8 to 12 h. Then, add 10 μL of CCK-8 solution and 90 μL of colorless DMEM medium to each well. After incubating for 45 min, measure the absorbance at a wavelength of 450 nm using a microplate reader to evaluate cell viability. The cell viability under a light intensity of 60 mW / cm 2 is shown by the purple columns in Figure 9 .

[0105] Figure 9The results showed that there was no significant change in the cell viability after incubation with samples at different concentrations compared with that of the control group, and the cell viability remained above 90%, indicating that TTCMSB NPs did not exhibit cytotoxicity in the concentration range of 160 μM and had good cell safety.

[0106] Figure 9 In the range of 60 mW / cm 2 light intensity, the inhibitory effect of a sample concentration of 4 μM on cell viability was approximately 50%; when the light sample concentration was 16 μM, the inhibition efficiency was approximately 80%; these results indicated that TTCMSB NPs could selectively target and kill MDA-MB-231 cells and had good PDT ability. Under light irradiation at a lower light intensity (60 mW / cm 2 ), TTCMSB NPs could effectively generate reactive oxygen species, induce apoptosis of a large number of MDA-MB-231 cells, and had a good effect on inhibiting tumor growth.

[0107] To further analyze the effects of TTCMSB NPs and light irradiation (60 mW / cm 2 light intensity) on cell viability, flow cytometry (FCM) was used to quantitatively analyze the changes in cell survival rate and apoptosis rate of MDA-MB-231 cells after incubation in an aqueous solution of TTCMSB NPs and under light irradiation conditions. The results were as Figure 10 shown. After incubating cells with sample solutions of different concentrations of TTCMSB NPs (0, 1 μM, 2 μM, 4 μM, 8 μM, and 16 μM) for 8 h, the inhibitory effect of a sample concentration of 8 μM on cell viability was approximately 50.8%; when the light sample concentration was 16 μM, the inhibition efficiency was approximately 71.8%; these results indicated that TTCMSB NPs could selectively target and kill MDA-MB-231 cells, had good PDT ability, could generate reactive oxygen species, and induce a large number of MDA-MB-231 cells to apoptosis.

[0108] Test Example 3

[0109] As an emerging non-invasive tumor treatment method, phototherapy is mainly divided into two methods: photodynamic therapy (PDT) and photothermal therapy (PTT). In PDT, the photodiagnostic agent generates singlet oxygen through energy conversion ( 1O2), which is the mechanism of type II PDT; while type I PDT forms free radicals (such as ·OH, ·O2ˉ, H2O2, etc.) through electron transfer to damage biological macromolecules. In addition to the photosensitizer and light source, type II PDT also relies on the participation of oxygen. However, the microenvironment of solid tumors usually shows a hypoxic state, which limits the effect of PDT. In contrast, type I PDT is not limited by oxygen supply and is therefore more commonly used in photodynamic tumor therapy. PTT can promote blood circulation by locally heating the irradiated area, thus alleviating the tumor hypoxia problem in PDT.

[0110] Determination of ROS In Vitro , and the specific steps are as follows:

[0111] (1) Determination of ·OH free radicals (HPF probe method): Add 2 μL of NPs or RB stock solution (1 mM) (i.e., working concentration 1 μM) and 5 μL of HPF stock solution (1 mM) (i.e., working concentration 1 μM) to 993 μL of PBS solution. After white light irradiation at different time intervals, record the fluorescence emission intensity of the mixed solution at 515 nm (EX: 495 nm);

[0112] (2) Determination of ·O2ˉ or H2O2 free radicals (DHR probe method): Add 2 μL of NPs or RB stock solution (1 mM) (i.e., working concentration 1 μM) and 10 μL of DHR123 stock solution (1 mM) (i.e., working concentration 1 μM) to 993 μL of PBS solution. After white light irradiation at different time intervals, record the fluorescence emission intensity of the mixed solution at 535 nm (EX: 505 nm);

[0113] (3) Total ROS determination (DCFH probe method): Mix 100 μL of 1 mM DCFH-DA stock solution with 400 μL of 0.01 M NaOH aqueous solution and place it at room temperature for 30 min to convert DCFH-DA to DCFH. Add 19.5 μL of PBS solution to make its final concentration 5 mM, then add nanoparticles and RB (working concentration of nanoparticles and RB is 1 μM) respectively. After white light irradiation at different time intervals, measure the fluorescence emission intensity of the mixed solution at 525 nm (EX: 485 nm); and calculate the photothermal conversion efficiency.

[0114] Photothermal conversion efficiency (η = (hs(T Max -T Surr )-Q Dis ) / (I(1 - 10 A635 ))

[0115] where h is the heat convection coefficient; s is the surface area of the material irradiated by light. T Max and T Surr represent the highest temperature and the ambient temperature respectively, QDis is the heat loss. I is the incident light power (0.8 W cm -2 ), and A 635 is the absorbance of the nanoparticles at 635 nm.

[0116] hs = (mC_water) / τ s

[0117] where m is the mass of the solution containing the photoactive material, C is the specific heat capacity of the solution (C_water = 4.2 J / (g·°C)), and τ s is the relevant time constant.

[0118] t = -τs ln(θ)

[0119] where θ is a dimensionless parameter called the driving force temperature.

[0120] Using a 635 nm laser (200 μM 0.8 W cm -2 ), the photothermal conversion efficiency of the aqueous solution of the nanoparticles was calculated.

[0121] The results are shown in Figure 11 b, 11c, 11e, 11f. TTCMSB NPs can generate reactive oxygen species (such as O2ˉ, ·OH) mainly for type I PDT treatment. Type I PDT is not limited by oxygen supply, can improve the photothermal conversion efficiency, and can generate a relatively high photothermal conversion efficiency (up to 40.5%) at a lower concentration and a lower light intensity (635 nm laser, 300 mW / cm 2 ). Combining phototherapy can effectively enhance the therapeutic effect and can be more efficiently used for killing tumor cells. It overcomes the problem of the prior art that a relatively high light intensity (800 mW / cm 2 ) is required to achieve the photothermal therapy effect (a relatively high light intensity will cause thermal damage or scalding).

[0122] The TTCMSB NPs system of the present invention can generate free radicals (such as O2ˉ, ·OH) that can support type I photodynamic therapy in addition to 1 O2. Combining the advantages of PDT and PTT, by generating singlet oxygen ( 1 O2) and free radicals (such as ·OH, ·O2ˉ), combining photothermal therapy can effectively enhance the therapeutic effect, and a good therapeutic effect can be achieved at a low light intensity (300 mW / cm 2 ). The present invention provides a more efficient phototherapy method. PTT can promote blood circulation by locally heating the irradiated area and further relieve the tumor hypoxia problem. Compared with the prior art, the present invention has significant advantages in generating reactive oxygen species and can significantly improve the phototherapy effect.

[0123] Test Example 4, Tumor Treatment Ability

[0124] Inject TTCMSB NPs into nude mice by in-situ injection to evaluate their tumor treatment ability in MDA-MB-231 tumor-bearing mice.

[0125] Method for constructing MDA-MB-231 tumor-bearing mice: Use BALB / c nude mice (purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.) at about four weeks old (about 16 - 20 g). Inject 100 μL of 5×10 6 MDA-MB-231 cells. After 3 - 4 weeks, if the tumor volume is about 100 mm 3 , it is considered that the MDA-MB-231 tumor-bearing mice are successfully constructed.

[0126] The specific steps of the evaluation method for the tumor treatment ability of the phototherapeutic agent are as follows:

[0127] (1) In vivo fluorescence / photoacoustic imaging; Use a PerkinElmer IVIS spectral imaging system to capture fluorescence images at different time points (0, 0.5 h, 2 h, 4 h, 8 h, 12 h, 24 h) after injection. During the photothermal therapy stage, select the time period from 1 to 10 min for imaging.

[0128] (2) When the tumor volume grows to about 50 mm 3 , randomly divide the tumor-bearing nude mice into four groups, with 6 animals in each group. The grouping is as follows: The first group receives PBS solution (100 μL) and is accompanied by laser irradiation (light intensity 300 mW / cm 2 ); The second group only receives PBS solution (100 μL) without laser irradiation; The third group receives TTCMSB NPs (100 μL) without laser irradiation; The fourth group receives TTCMSB NPs (100 μL) and is combined with laser irradiation (light intensity 300 mW / cm 2 ).

[0129] (3) 1 h after injection, use a laser (light intensity 300 mW / cm 2 ) to irradiate the tumor for 10 min. The treatment is performed every two days for 2 - 3 times for 14 consecutive days, and the tumor size and animal body weight are recorded every other day during this period.

[0130] (4) The tumor size is calculated using the formula: Tumor volume = 0.5 × tumor length × tumor width.

[0131] (5) In terms of statistical analysis, the data are presented in the form of mean ± standard deviation (Mean ± S.D.). ANOVA variance analysis was performed using GraphPad Prism 8 software to determine whether the differences between the data are statistically significant. The criteria for determination are: p < 0.05, p < 0.01, and p < 0.001 indicate that the differences are statistically significant.

[0132] Some of the results are as Figure 12 , TTCMSB NPs can not only achieve near-infrared I / II fluorescence imaging ability ( Figure 12 a / b), indicating that TTCMSB NPs have good fluorescence imaging ability; in addition, after injecting TTCMSB NPs into mice, laser irradiation (300 mW / cm 2 ) was performed 1 h later. When the laser irradiation lasted for 1 min, the temperature at the tumor site of the experimental group of mice rapidly rose to 50 °C at a lower light intensity (300 mW / cm 2 ), and further rose to 60 °C after 5 min. In the control group, even after receiving PBS injection and laser irradiation under the same conditions, the temperature at the tumor site only reached 45.1 °C after 5 min, indicating that TTCMSB NPs have a significant photothermal effect in vivo (the photothermal conversion efficiency is 40.5%), can be effectively converted into heat energy, thereby exerting a killing effect on tumor cells, overcoming the problem that the existing phototherapeutic agents require a higher light intensity (such as 800 mW / cm 2 ) to achieve the phototherapeutic effect (because the existing phototherapeutic agents mainly rely on type II photodynamic therapy (PDT) that requires oxygen to form reactive oxygen species (ROS), and need a higher photothermal effect (i.e., a higher light intensity) for oxygen supply, with a lower photothermal conversion efficiency, and a higher light intensity will cause thermal damage or scalding, which is not photothermal and photodynamic therapy).

[0133] After the treatment, the tumor volume of the mice was measured and histological evaluation was performed. The results are as Figure 13 , Figure 13 a shows that in the mice treated with the combination of TTCMSB NPs and laser irradiation, the tumor volume was significantly reduced, even completely ablated, and no tumor recurrence was observed during the 14-day observation period, only scars remained at the original tumor site. In addition, compared with the control group, the body weight of the experimental group of mice did not show a significant change, indicating that TTCMSB NPs have low toxicity (see Figure 13 b).

[0134] The results of the above examples show that the aggregation-induced emission material provided by the present invention has a therapeutic effect mainly based on type I photodynamic therapy and can be used in the application of photothermal-type I photodynamic synergistic ablation therapy drugs for malignant tumors.

[0135] Meanwhile, the present invention also compared the photothermal temperature changes of TTCMSB NPs and TPTMSB NPs, and the results are shown in Table 1 and Figure 14 .

[0136] Table 1 Photothermal temperature changes of nanoparticles in vivo

[0137] 0 min 1 min 2 min 3 min 4 min 5 min 6 min 7 min 8 min 9 min 10 min PBS (°C) 36.4 39.1 40.4 40.9 41 41 41.3 41.6 42 41.3 41 TPTMSB (°C) 36.4 45.3 48.4 50.8 52.1 53.1 54.1 54.3 54.5 54.3 54.1 TTCMSB (°C) 36.3 50 54.2 59.5 60.1 60 59.7 60.1 60.8 60.7 61

[0138] The results in Table 1 show that compared with the PBS group, the temperatures of both the TPTMSB NPs group and the TTCMSB NPs group were significantly increased.

[0139] Table 1 and Figure 14 The results show that at the 3-minute time point, compared with the PBS control group, the temperature of the TPTMSB NPs group rose to 50.8 °C, while the temperature of the TTCMSB NPs group increased to 59.5 °C; at the 6-minute time point, compared with the PBS control group, the temperature of the TPTMSB NPs group further rose to 54.1 °C, while the temperature of the TTCMSB NPs group rose to 59.7 °C; at the 9-minute time point, compared with the PBS control group, the temperature of the TPTMSB NPs group stabilized at 54.3 °C, while the temperature of the TTCMSB NPs group significantly rose to 60.7 °C. That is, compared with the TPTMSB NPs group, the TTCMSB NPs group had a faster temperature rise in a short time and had a better therapeutic effect.

[0140] In photothermal therapy (PTT), the rapid rise of temperature in a short time has a significant impact on the therapeutic effect. First of all, the rapid temperature rise can quickly reach a temperature sufficient to destroy tumor cells, usually above 42 °C, which can cause cell death in a relatively short time. At the same time, the rapid temperature rise helps to reduce the treatment time, improve the treatment efficiency, and may reduce the thermal damage to surrounding normal tissues. In addition, the rapid temperature rise can also increase the permeability of tumor tissues, help the absorption and diffusion of drugs, and enhance the therapeutic effect.

[0141] The phototherapeutic agent of the present invention (such as TTCMSB NPs) mainly combines type I photodynamic therapy (PDT) that does not require oxygen with photothermal therapy. Because type I photodynamic therapy (PDT) can rapidly generate reactive oxygen species without relying on oxygen, the photodiagnostic agent of the present invention (such as TTCMSB NPs) has a high reactive oxygen species generation ability and photothermal conversion efficiency under low light intensity (300 mW / cm 2 ) conditions, as well as the combination of photothermal therapy (PTT) and rapid heating to a relatively high temperature (56 - 61 °C, even up to 59 - 61 °C), showing excellent photothermal conversion efficiency. And the short-time temperature rise has an important impact on PTT treatment, which can effectively achieve the purpose of killing tumors in a short time, thereby improving the treatment efficiency and efficacy.

[0142] III-1 of the Present Invention The compounds shown in III-2, IV-1, IV-2, and IV-3 also mainly rely on oxygen-independent type I photodynamic therapy (PDT) combined with photothermal therapy, and have a high photothermal conversion efficiency.

[0143] In the present invention, the introduction of sulfonic acid groups significantly enhances the water solubility of the molecules, thereby improving the solubility and reactivity of these molecules in vivo. This enhanced solubility is generally associated with better biocompatibility. In addition, as a hydrogen bond acceptor, the sulfonic acid group further enhances the biological activity of the molecules by establishing additional hydrogen bond interactions with biological targets.

[0144] The results of the above examples show that the amphiphilic aggregation-induced emission materials provided by the present invention have a therapeutic effect mainly based on type I photodynamic combined with photothermal therapy, and can be used for multimodal diagnostic detection of malignant tumors and optical imaging-guided photothermal-type I photodynamic synergistic therapy.

[0145] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. An amphiphilic AIE phototheranostic agent with the synergistic effect of photothermal and type I reactive oxygen species, which comprises a compound shown by the structural formula as formula I, or a pharmaceutically acceptable salt thereof, a solvate thereof and a tautomer thereof; Among them, R is: n linked groups, where 1 ≤ n ≤ 5, and the wavy line represents the connection point; X is selected from one or more of O, S and Se.

2. The amphiphilic AIE phototheranostic agent according to claim 1, wherein 1 ≤ n ≤ 3, optionally n = 2 or 3.

3. The amphiphilic AIE-based photodiagnostic agent according to claim 1 or 2, wherein It comprises one or more of the following compounds shown by the structural formula, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a tautomer thereof:

4. The amphiphilic AIE phototheranostic agent according to any one of claims 1 to 3, characterized in that, It is a nanotheranostic agent, optionally prepared from the compound shown by formula I through a self-assembly technique.

5. A method for preparing the amphiphilic AIE photodiagnostic agent according to any one of claims 1 to 4, characterized in that, It comprises the following steps: in a solvent, under the action of a basic substance, the compounds shown by formula A and formula B generate the compound shown by formula I:

6. The preparation method according to claim 5, characterized in that, The solvent is selected from one or more of N,N-dimethylformamide, dimethyl sulfoxide, toluene, o-xylene, dichloromethane and chloroform; And / or, the basic substance is selected from one or more of pyridine, triethylamine, potassium carbonate and sodium carbonate; And / or, the reaction is carried out in an anaerobic atmosphere; And / or, the reaction temperature is 80 - 200 °C, optionally 100 - 200 °C, optionally 120 - 180 °C, optionally 140 - 160 °C, optionally 150 - 160 °C; And / or, the reaction time is 10 - 18 h, optionally 12 - 18 h; And / or, the molar ratio of the compounds shown by formula A and formula B is 1:0.9 - 1.5, optionally 1:1 - 1.5, optionally 1:1 - 1.

2.

7. The preparation method according to claim 5 or 6, characterized in that, Preparing the compound shown by formula I into nanoparticles: dissolving the compound shown by formula I in an organic solvent and deionized water, and obtaining the nanoparticles by ultrasonic treatment, dialysis and filtration; Optionally, the organic solvent is selected from one or more of DMSO, dimethyl sulfoxide and tetrahydrofuran; Optionally, the ultrasonic treatment time is 2 - 40 min, optionally 30 - 40 min; Optionally, the ultrasonic power is 20 - 300 W; Optionally, dialysis is carried out in deionized water, optionally, the deionized water is changed once every 6 - 10 h, and optionally, dialysis is carried out for 24 - 56 h; Optionally, the pore size of filtration is 0.1 - 0.8 μM, optionally 0.22 μM.

8. The preparation method according to any one of claims 5 to 7, characterized in that, The preparation method of the compound shown by formula B comprises: reacting 1,3-propane sultone and 2-methylbenzothiazole in a solvent to obtain the compound shown by formula B; Optionally, in the preparation of the compound shown by formula B, the solvent is toluene; Optionally, in the preparation of the compound shown by formula B, the reaction is carried out in an inert gas atmosphere; Optionally, the reaction temperature is 45 - 120 °C, optionally 80 - 120 °C, optionally 100 - 120 °C, and the reaction time is 6 - 10 h; Optionally, the molar ratio of 1,3-propane sultone and 2-methylbenzothiazole is 1:0.9 - 1.5, optionally 1:1 - 1.5, optionally 1:1 - 1.

2.

9. Use of the amphiphilic AIE phototheranostic agent according to any one of claims 1 to 4, or the amphiphilic AIE phototheranostic agent prepared by the preparation method according to any one of claims 5 to 8, in the preparation of an aggregation-induced emission type phototheranostic agent, or a product targeting tumor cell lysosomes, or an anti-tumor product combining type I photodynamic therapy and photothermal therapy, or a tumor fluorescence-photoacoustic-photothermal imaging-guided photothermal-photodynamic synergistic tumor therapy product, or a phototheranostic agent product mainly based on type I photodynamic therapy and photothermal therapy.

10. Use of the amphiphilic AIE phototheranostic agent according to any one of claims 1 to 4, or the amphiphilic AIE phototheranostic agent prepared by the preparation method according to any one of claims 5 to 8, in the preparation of a photodynamic-photothermal combined anti-tumor phototheranostic agent mediated by near-infrared region I and region II fluorescence imaging, photoacoustic imaging, and photothermal imaging, or an anti-tumor product by photothermal therapy, or a photothermal-type I photodynamic synergistic ablation therapy drug for malignant tumors. Optionally, the amphiphilic AIE photo-theranostic agent can reach a photothermal temperature of 56-61 °C, optionally 59-61 °C, under a light intensity of 300 mW / cm 2 2 Optionally, the use concentration of the amphiphilic AIE phototheranostic agent is 0.1 to 20 μM, optionally 1 to 16 μM, optionally 0.5 to 2 μM, and optionally 1 to 3 μM.

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