Near-infrared two-region aggregation-induced emission probe, nanoparticle, and preparation method and application of near-infrared two-region aggregation-induced emission probe and nanoparticle

Abstract

The invention discloses a near-infrared two-region aggregation-induced emission probe, nanoparticles, a preparation method and application, and relates to the technical field of fluorescent molecular probes. The structural formula of the near-infrared two-region aggregation-induced emission probe is shown in the specification, wherein X1, X2 and Y are respectively and independently selected from one of O, S, Se and Te; r1, R2 and R3 are respectively and independently selected from one of H, F, cyano, trifluoromethyl and saturated alkane. The probe provided by the invention has relatively high luminous efficiency, photo-thermal efficiency and active oxygen generation capability in an aggregation state, and can realize fluorescence, photo-acoustic and photo-thermal multi-mode imaging and photo-thermal / photodynamic synergistic treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fluorescent molecular probe, in particular to a near-infrared two-region aggregation-induced emission probe, a nanoparticle and a preparation method and application thereof. BACKGROUND

[0002] As a major disease threatening human health, precise diagnosis and efficient treatment of tumors have always been the core challenge in the medical field. Traditional diagnosis and treatment methods have significant limitations in terms of tissue penetration depth, spatial and temporal resolution, and treatment specificity, making it difficult to meet the urgent need for high-precision diagnosis and treatment integration in clinical practice. In recent years, optical diagnosis and treatment technology based on the near-infrared two-region (NIR-II, 1000-1700 nm) optical window has opened up a new path for precise diagnosis and treatment of tumors due to its advantages such as deep tissue penetration, high spatial and temporal resolution, low biological background interference, and multiple treatment modalities. Among them, organic near-infrared two-region probes with aggregation-induced emission (AIE) characteristics are particularly eye-catching. Their unique anti-quenching effect can effectively overcome the problem of fluorescence quenching caused by aggregation in biological environments (ACQ), and they also exhibit significant advantages in photothermal conversion efficiency and reactive oxygen species generation capacity. However, the existing near-infrared two-region aggregation-induced emission probe molecules have few structural types. Therefore, how to construct novel near-infrared two-region aggregation-induced emission probe molecules through molecular engineering and achieve efficient fluorescence, photoacoustic, and photothermal multi-modal imaging and photothermal / photodynamic synergistic therapy of aggregation-induced emission probes in the near-infrared two-region is a scientific problem that needs to be solved urgently.

[0003] Therefore, the prior art still needs to be improved and developed. SUMMARY

[0004] Based on the deficiencies of the prior art described above, the purpose of the present application is to provide a near-infrared two-region aggregation-induced emission probe, a nanoparticle and a preparation method and application thereof, aiming to provide a near-infrared two-region aggregation-induced emission probe with novel structure, high luminescent efficiency, photothermal efficiency and reactive oxygen species (ROS) generation capacity, to solve the problem of few structural types of existing near-infrared two-region aggregation-induced emission probe molecules.

[0005] The technical solution of the present application is as follows: In a first aspect of the present application, a near-infrared two-region aggregation-induced emission probe is provided, wherein the structural formula of the near-infrared two-region aggregation-induced emission probe is: ; wherein X1, X2 and Y are each independently selected from one of O, S, Se, Te; R1, R2 and R3 are each independently selected from one of H, F, cyano, trifluoromethyl, saturated alkyl.

[0006] Optionally, the saturated alkyl group comprises one of methyl, tert-butyl, and octyl.

[0007] In a second aspect, the present application provides a preparation method of the near-infrared two-region aggregation-induced emission probe according to the present application, wherein the preparation method comprises the following steps: After the reaction of and , the near-infrared two-region aggregation-induced emission probe is obtained.

[0008] Optionally, the preparation method of the compound of formula II comprises the following steps: After the reaction of , hydrated ruthenium trichloride, and sodium periodate, the compound of formula II is obtained; and / or, The preparation method of the compound of formula I comprises the following steps: After the reaction of , iron powder, and acetic acid, the compound of formula I is obtained.

[0009] Optionally, the preparation method of the compound of formula IV comprises the following steps: After the reaction of and , the compound of formula IV is obtained.

[0010] Optionally, the preparation method of the compound of formula V comprises the following steps: After the reaction of and , the compound of formula V is obtained. ; After the reaction of and N - bromo-succinimide, the compound of formula V is obtained.

[0011] Optionally, the preparation method of the compound of formula VI comprises the following steps: After the reaction of and , the compound of formula VI is obtained. ; After the reaction of , 4,4'-di-tert-butyl-2,2'-bipyridine, [Ir(COD)(OMe)]2, and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane, the compound of formula VI is obtained.

[0012] In a third aspect of the present application, a nanoparticle is provided, wherein the nanoparticle comprises a near-infrared two-region aggregation-induced emission probe and an amphiphilic carrier coated on the surface of the near-infrared two-region aggregation-induced emission probe; the near-infrared two-region aggregation-induced emission probe is the near-infrared two-region aggregation-induced emission probe as described above or prepared by the preparation method as described above.

[0013] In a fourth aspect of the present application, a preparation method of the nanoparticle as described above is provided, comprising the following steps: The near-infrared two-region aggregation-induced emission probe and the amphiphilic carrier are added into water, and then the nanoparticle is obtained by sequentially performing ultrasonic treatment, dialysis and ultrafiltration concentration.

[0014] In a fifth aspect of the present application, the near-infrared two-region aggregation-induced emission probe as described above, the near-infrared two-region aggregation-induced emission probe prepared by the preparation method as described above, the nanoparticle as described above or the nanoparticle prepared by the preparation method as described above are applied to the preparation of a multimodal diagnosis and treatment integrated nanodrug.

[0015] Beneficial effects: the present application adopts the design principle of "skeleton distortion and molecular rotor" to obtain a multifunctional light diagnosis and treatment probe with imaging and treatment functions, which can be used in the field of tumor multimodal diagnosis and treatment integration. Specifically, the present application takes pyrene bisphenazene thiazole with strong electron-withdrawing units as an acceptor (A), an ortho-substituted thiophene or furan as a first π-bridge, a multi-position substituted benzothiophene or benzofuran as a second π-bridge, and an aromatic amine compound as a donor (D) to obtain a D-π-A-π-D type near-infrared two-region aggregation-induced emission diagnosis and treatment probe with position isomerization of π-bridge. Such probes have high luminescent efficiency, photothermal efficiency and active oxygen (ROS) generation capacity in an aggregated state, and can realize fluorescence, photoacoustic, photothermal multimodal imaging and photothermal / photodynamic synergistic therapy. At the same time, the introduction of rigid conjugated units benzothiophene or benzofuran as the second π-bridge can conveniently change the position of the donor to regulate the photophysical behavior and energy dissipation path of the probe, and realize the fine regulation of the imaging and treatment performance of such probes. b ]thiophene or benzothiophene as the second π-bridge can conveniently change the position of the donor to regulate the photophysical behavior and energy dissipation path of the probe, and realize the fine regulation of the imaging and treatment performance of such probes. b b ]thiophene or benzothiophene as the second π-bridge can conveniently change the position of the donor to regulate the photophysical behavior and energy dissipation path of the probe, and realize the fine regulation of the imaging and treatment performance of such probes. b BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The structural formula of the near-infrared two-region aggregation-induced emission probe.

[0017] Figure 2 The nuclear magnetic resonance hydrogen spectrum of compound 5BT-PBPT prepared in Example 1 in deuterated chloroform. ​​

[0018] Figure 3 The image shows the carbon NMR spectrum of compound 5BT-PBPT prepared in Example 1 in deuterated chloroform.

[0019] Figure 4 The mass spectrum of compound 5BT-PBPT prepared in Example 1 is shown.

[0020] Figure 5 The image shows the 1H NMR spectrum of compound 6BT-PBPT prepared in deuterated chloroform in Example 2.

[0021] Figure 6 The image shows the carbon NMR spectrum of compound 6BT-PBPT prepared in Example 2 in deuterated chloroform.

[0022] Figure 7 The mass spectrum of compound 6BT-PBPT prepared in Example 2 is shown.

[0023] Figure 8 The absorption spectra of compounds 5BT-PBPT and 6BT-PBPT prepared in Examples 1 and 2 in tetrahydrofuran are shown.

[0024] Figure 9 The graphs show the aggregation-induced emission curves of compounds 5BT-PBPT and 6BT-PBPT prepared in Examples 1 and 2 in a mixed solution of tetrahydrofuran and water.

[0025] Figure 10 The photoluminescence patterns of the 5BT-PBPT NPs and 6BT-PBPT NPs prepared in Example 3 are shown.

[0026] Figure 11 The image shows the photothermal conversion efficiency of the 5BT-PBPT NPs prepared in Example 3.

[0027] Figure 12 The image shows the photothermal conversion efficiency of the 6BT-PBPT NPs prepared in Example 3.

[0028] Figure 13 The graph shows the ROS generation capabilities of the 5BT-PBPT NPs and 6BT-PBPT NPs prepared in Example 3.

[0029] Figure 14 The image shows the dark toxicity and photothermal-photodynamic synergistic killing effect of the 5BT-PBPT NPs prepared in Example 3 on 4T1 cells.

[0030] Figure 15This is a fluorescence / photoacoustic / photothermal synergistic imaging effect of the 5BT-PBPT NPs prepared in Example 3 on in situ breast cancer.

[0031] Figure 16 The image shows the photothermal-photodynamic synergistic therapeutic effect of the 5BT-PBPT NPs prepared in Example 3 on in situ breast cancer. Detailed Implementation

[0032] This invention provides a near-infrared II region aggregation-induced emission probe, nanoparticles, preparation method, and application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0033] Unless otherwise defined, 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 invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0034] If the embodiments of the present invention involve descriptions such as "first" or "second", such descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated.

[0035] This invention provides a near-infrared II region aggregation-induced emission probe, wherein, as... Figure 1 As shown, the structural formula of the near-infrared II region aggregation-induced emission probe is: ; X1, X2, and Y are each independently selected from one of O, S, Se, and Te; R1, R2, and R3 are each independently selected from one of H, F, cyano (-CN), trifluoromethyl (-CF3), and saturated alkane group (selecting these groups for R1, R2, and R3 can make the donor unit have good electron-donating ability, and make the material have good solubility and biocompatibility).

[0036] This invention employs a "skeleton twisting, molecular rotor" design principle to obtain a class of multifunctional phototherapy probes that combine imaging and therapy, applicable to the field of integrated multimodal tumor diagnosis and treatment. Specifically, this invention uses the strong electron-withdrawing unit pyrene-bisphenol azidothiadiazole as the acceptor (A), and ortho-substituted thiophene or furan as the first π-bridge, allowing for multi-positional substitution of benzo[…]. b ]Thiophene or benzo[ bUsing furan and other compounds as the second π-bridge and aromatic amine compounds as donors (D), a class of D-π-A-π-D type near-infrared two-region aggregation-induced emission diagnostic and therapeutic probes were obtained through positional isomerization of the π-bridge. These probes exhibit high luminescence efficiency, photothermal efficiency, and reactive oxygen species (ROS) generation capacity in the aggregated state, enabling multimodal imaging via fluorescence, photoacoustic, and photothermal methods, as well as synergistic photothermal / photodynamic therapy. Simultaneously, the rigid conjugated unit benzo[…] b ]Thiophene or benzo[ b The introduction of furan as a second π-bridge allows for convenient modification of the donor's position to regulate the probe's photophysical behavior and energy dissipation path, enabling precise control over the imaging and therapeutic performance of such probes.

[0037] In some embodiments, the saturated alkane group includes methyl (-CH3), tert-butyl (-C(CH3)3), octyl (-C8H) 17 One of them.

[0038] This invention also provides a method for preparing the near-infrared II aggregation-induced emission probe as described above, wherein the method for preparing the near-infrared II aggregation-induced emission probe includes the following steps: Synthesis Route 1:

[0039] Among them, X1, X2, Y, R1, R2 and R3 in the structural formulas of the compounds shown in Formula I and Formula II are the same as X1, X2, Y, R1, R2 and R3 in the structural formula of the near-infrared II region aggregation-induced emission probe mentioned above.

[0040] Following the above synthetic route 1, the compound shown in Formula I and the compound shown in Formula II are reacted to obtain the near-infrared II region aggregation-induced emission probe.

[0041] The method for preparing near-infrared II aggregation-induced emission probes provided by this invention has a mature synthetic route and reaction type, readily available raw materials, strong modifiability, and good biocompatibility, providing a new approach for multimodal phototherapy research on diseases such as tumors.

[0042] In one specific embodiment, the step of reacting the compound shown in Formula I with the compound shown in Formula II to obtain the near-infrared II aggregation-induced emission probe specifically includes: Following synthetic route 1 above, a mixed solution of CHCl3 and AcOH (where the volume ratio of CHCl3 to AcOH is (1~10):1, for example, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, etc.) was added to a mixture of the compound shown in Formula I and the compound shown in Formula II (the molar ratio of the compound shown in Formula I and the compound shown in Formula II is 2:1). The mixture was heated to 80°C under stirring for 24 hours. After cooling to room temperature, the reaction mixture was poured into water and extracted three times with dichloromethane. The combined organic layers were dried over anhydrous Na2SO4, concentrated by air separation, and the crude product was purified by silica gel column chromatography using petroleum ether and dichloromethane as eluents to obtain the near-infrared II aggregation-induced emission probe.

[0043] In some embodiments, the preparation method of the compound represented by Formula II includes the following steps: Synthesis Route 2:

[0044] in, R2 in the structural formula is the same as R2 in the near-infrared II region aggregation-induced emission probe structural formula above; ACN is acetonitrile (the same below), and DCM is dichloromethane (the same below). Following the above synthetic route 2, at room temperature, Dissolved in a mixed solution of acetonitrile and dichloromethane (acetonitrile to dichloromethane volume ratio 1:1), catalytically added ruthenium trichloride hydrate (RuCl3·3H2O) in a single batch, and under stirring, sodium periodate (NaIO4) solid added in batches (while ensuring...). The mixture was prepared by mixing RuCl3·3H2O and NaIO4 in a molar ratio of 1:0.06:40, then heating to 35°C and stirring continuously for 3 hours. After cooling to room temperature, it was extracted with dichloromethane, the organic phase was dried over anhydrous NaSO4, and the crude product was purified by silica gel column chromatography to obtain the compound shown in Formula II.

[0045] In some embodiments, the preparation method of the compound represented by Formula I includes the following steps: Synthesis Route 3:

[0046]

[0047] Among them, X1, X2, Y, R1 and R3 in the structural formulas of compounds shown in Formula VI, Formula V and Formula IV are the same as X1, X2, Y, R1 and R3 in the structural formula of the near-infrared II aggregation-induced emission probe mentioned above.

[0048] Following the above synthetic route 3, the compound shown in formula V is reacted with the compound shown in formula VI to obtain the compound shown in formula IV. The compound shown in Formula IV, iron powder, and acetic acid were mixed and reacted to obtain the compound shown in Formula I.

[0049] In some specific embodiments, the preparation method of the compound represented by Formula I includes the following steps: Following synthetic route 3 above, under a N2 atmosphere, the compounds shown in Formula VI, Formula V, potassium carbonate, and Pd(PPh3)4 were mixed in a molar ratio of 2:1:(1~3):(0.01~0.06. The mixture of these compounds, a 2M aqueous solution of potassium carbonate, and Pd(PPh3)4 was then added, followed by mixing with 1,4-dioxane. The mixture was stirred overnight (12 h) at 80 °C. After cooling to room temperature, the solvent was removed under reduced pressure, the product was redissolved in dichloromethane, washed with water, dried over anhydrous Na2SO4, and the crude product was purified by silica gel column chromatography, eluting with dichloromethane and petroleum ether to obtain the compound shown in Formula IV. Under a nitrogen atmosphere, the compound of formula IV and excess iron powder (the molar ratio of the compound of formula IV to iron powder is 1:(10~20), for example, 1:10, 1:12, 1:15, 1:18 or 1:20, etc.) were added to a round-bottom flask containing acetic acid. The mixture was heated at 80°C and stirred vigorously for 4 hours. During this period, a large amount of solid precipitated from the system. After the reactants were consumed, the reaction was cooled to room temperature and an appropriate amount of distilled water was added. The crude product was filtered through diatomaceous earth, redissolved in dichloromethane, and washed with water. The organic layer was dried with anhydrous Na2SO4, the solvent was removed under reduced pressure, and the mixture was recrystallized from methanol / dichloromethane to obtain the compound of formula I.

[0050] For example, the molar ratio of the compound shown in Formula VI, the compound shown in Formula V, potassium carbonate and Pd(PPh3)4 can be 2:1:1:0.01, 2:1:1:0.03, 2:1:1:0.06, 2:1:3:0.01, 2:1:3:0.03 or 2:1:3:0.06, etc.

[0051] In some embodiments, the preparation method of the compound represented by formula V includes the following steps: Synthesis Route 4:

[0052]

[0053] In the compounds shown in Formula VIII, Formula IX, and Formula VII, X2, Y, and R1 are the same as X2, Y, and R1 in the near-infrared II aggregation-induced emission probe structure described above; NBS represents N - Bromosuccinimide (hereinafter the same), DMF represents N,N-dimethylformamide (hereinafter the same). Following the above synthetic route 4, the compound shown in formula VIII was reacted with the compound shown in formula IX to obtain the compound shown in formula VII; The compound shown in equation VII and N After reacting with bromosuccinimide, the compound shown in formula V is obtained.

[0054] In some specific embodiments, the preparation method of the compound shown in Formula V includes the following steps: Following synthetic route 4 above, under a nitrogen atmosphere, the compound shown in formula VIII, the compound shown in formula IX, and Pd(PPh3)2Cl2 were added to toluene in a molar ratio of 1:2:(0.01~0.06), stirred, and refluxed overnight (i.e., 110℃, 12h). After cooling to room temperature, a saturated KF solution (saturated Karl Fischer solution) was added, the reaction mixture was poured into water and extracted with dichloromethane, the organic layer was dried over anhydrous Na2SO4, concentrated, and the residue was purified by column chromatography, eluted with petroleum ether and dichloromethane, to obtain the compound shown in formula VII. In a light-protected environment, the compound shown in Formula VII and N -bromosuccinimide (the compound shown in formula VII and N The 1:2 molar ratio of 1-bromosuccinyl ester was added to a mixed solution of acetonitrile and N,N-dimethylformamide (volume ratio of acetonitrile and N,N-dimethylformamide was 2:1). The mixture was heated and stirred at 60°C for 4 hours, cooled to room temperature, and the reaction was quenched with water. The reaction solution was poured into saturated brine and extracted multiple times with dichloromethane. The organic phase was dried over anhydrous Na2SO4 and then evaporated to dryness. The crude product was separated and purified by silica gel using dichloromethane / petroleum ether as the eluent to obtain the compound shown in Formula V.

[0055] For example, the molar ratio of the compound shown in Formula VIII, the compound shown in Formula IX, and Pd(PPh3)2Cl2 can be 1:2:0.01, 1:2:0.02, 1:2:0.03, 1:2:0.04, 1:2:0.05, or 1:2:0.06, etc.

[0056] In some embodiments, the preparation method of the compound shown in VI includes the following steps: Synthesis Route 5:

[0057]

[0058] Among them, R3 and X1 in Formula XI, Formula XII, and Formula X are the same as R3 and X1 in the near-infrared two-region aggregation-induced emission probe structure above; t BuONa represents sodium tert-butoxide (hereinafter the same). t Bu3PHBF4 represents tri-tert-butylphosphine tetrafluoroborate (the same below), dtbpy represents 4,4'-di-tert-butyl-2,2'-bipyridine (the same below), Hbpin represents 4,4,5,5-tetramethyl-1,3,2-dioxoborane (the same below); [Ir(COD)(OMe)]2 represents methoxy(cyclooctadiene)iridium dimer (the same below).

[0059] Following the above synthetic route 5, under a N2 atmosphere, formulas XI, XII, Pd(OAc)2, sodium tert-butoxide, tritert-tert-butylphosphine tetrafluoroborate (molar ratio of 1:1:(0.03~0.06):3:(0.03~0.06)) and toluene were added to a sealed tube. After heating and refluxing at 110°C for 12 h, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth and washed with CH2Cl2. The mixed organic solvents were removed by vacuum rotary evaporation. The crude product was purified by silica gel column chromatography with petroleum ether as the eluent to obtain the compound shown in formula X. Under a nitrogen atmosphere, the compound of formula X, 4,4'-di-tert-butyl-2,2'-bipyridine, [Ir(COD)(OMe)]2, and 4,4,5,5-tetramethyl-1,3,2-dioxoborane (molar ratio 1:(0.1~0.3):(0.01~0.05):(2~5)) and tetrahydrofuran were mixed and heated under reflux for 6 h. After cooling to room temperature, the reaction mixture was evaporated to dryness under reduced pressure, and the crude product was recrystallized from n-hexane to give the compound of formula VI.

[0060] For example, the molar ratios of formula XI, formula XII, Pd(OAc)2, sodium tert-butoxide, and tritert-tert-butylphosphine tetrafluoroborate can be 1:1:0.03:3:0.03, 1:1:0.03:3:0.06, 1:1:0.04:3:0.03, 1:1:0.04:3:0.06, 1:1:0.06:3:0.03, or 1:1:0.06:3:0.06, etc.

[0061] The molar ratio of the compound shown in Formula X, 4,4'-di-tert-butyl-2,2'-bipyridine, [Ir(COD)(OMe)]2 and 4,4,5,5-tetramethyl-1,3,2-dioxoborane can be 1:0.1:0.01:2, 1:0.1:0.01:5, 1:0.1:0.05:2, 1:0.1:0.05:5, 1:0.3:0.01:2, 1:0.3:0.01:5, 1:0.3:0.05:2 or 1:0.3:0.05:5, etc.

[0062] This invention also provides a nanoparticle, wherein the nanoparticle comprises a near-infrared II aggregation-induced emission probe and an amphiphilic carrier coating the surface of the near-infrared II aggregation-induced emission probe; the near-infrared II aggregation-induced emission probe is the near-infrared II aggregation-induced emission probe described in this invention embodiment or a near-infrared II aggregation-induced emission probe prepared by the preparation method described in this invention embodiment. In this embodiment, coating with an amphiphilic carrier can significantly improve the water solubility and biocompatibility of this type of hydrophobic near-infrared II aggregation-induced emission probe, which helps to evaluate the phototherapy performance of the probe at both in vitro and in vivo levels.

[0063] This invention also provides a method for preparing the nanoparticles described above, comprising the following steps: Near-infrared II aggregation-induced emission probe and amphiphilic carrier were added to water, and the mixture was subjected to ultrasonication, dialysis, and ultrafiltration concentration in sequence to obtain the nanoparticles.

[0064] The embodiments of the present invention also provide the near-infrared II aggregation-induced emission probe as described above, the near-infrared II aggregation-induced emission probe prepared by the preparation method as described above, and the nanoparticles as described above or prepared by the preparation method as described above, in the preparation of multimodal therapeutic nanomedicines.

[0065] The present invention will be further described below through specific embodiments.

[0066] Unless otherwise specified, the raw materials used in the following embodiments are all commercially available products.

[0067] In the following embodiments, the synthesis route of the near-infrared II aggregation-induced emission probe is as follows:

[0068]

[0069]

[0070]

[0071]

[0072]

[0073] Among them, compounds e1, f1, h1, j1, and 5BT-PBPT In compound d1, Br is located at position 5 of its benzene ring; in compounds e2, f2, h2, j2, and 6BT-PBPT... The Br in compound d2 is located at position 6 on its benzene ring.

[0074] Example 1 Following the above synthetic route, the preparation method of the near-infrared II aggregation-induced emission probe, namely compound 5BT-PBPT, includes the following steps: (1) Under light-protected conditions, compound a (4.88 mmol, readily available for purchase) and N - Bromosuccinimide (9.76 mmol) was added to acetonitrile (12 mL) and N , N In a mixed solution of dimethylformamide (24 mL), the mixture was heated and stirred at 60 °C for 4 h, then cooled to room temperature. The reaction was quenched with water, and the reaction solution was poured into saturated brine. The mixture was extracted multiple times with dichloromethane. The organic phase was dried over anhydrous Na2SO4 and then evaporated to dryness. The crude product was separated and purified by silica gel using dichloromethane / petroleum ether as the eluent to obtain an orange solid, namely compound b (3.5 g, 92.8%).

[0075] (2) Under N2 atmosphere, compound c (25.34 mmol), compound d1 (25.34 mmol), Pd(OAc)2 (0.76 mmol), sodium tert-butoxide (76.02 mmol), tritert-butylphosphine tetrafluoroborate (1.52 mmol) and toluene (150 mL) were added to a sealed tube and heated under reflux at 110°C for 12 h. After cooling to room temperature, the reaction mixture was filtered through diatomaceous earth and washed with CH2Cl2. The mixed organic solvent was removed by vacuum rotary evaporation. The crude product was purified by silica gel column chromatography with petroleum ether as the eluent to obtain a colorless solid, namely compound e1 (7.4 g, 88.62%).

[0076] (3) Under N2 atmosphere, compound e1 (9.56 mmol), 4,4'-di-tert-butyl-2,2'-bipyridine (0.96 mmol) and [Ir(COD)(OMe)]2 (0.38 mmol) were added to tetrahydrofuran (80 mL), and then 4,4,5,5-tetramethyl-1,3,2-dioxoborane (47.81 mmol) was added. The mixture was heated under reflux for 6 h. The reaction color changed from red to dark black. After cooling to room temperature, the reaction mixture was evaporated to dryness under reduced pressure. The crude product was recrystallized from n-hexane to give a gray solid, namely compound f1 (3.0 g, 68.90%).

[0077] (4) Under N2 atmosphere, compound f1 (2.04 mmol), compound g (1.02 mmol), potassium carbonate aqueous solution (2M, 1.53 mL) and Pd(PPh3)4 (0.021 mmol) were mixed, and then 1,4-dioxane (40 mL) was added and mixed. The mixture was stirred at 80 °C overnight (i.e., 12 h). After cooling to room temperature, the solvent was removed under reduced pressure, dichloromethane was redissolved, washed with water, dried with anhydrous Na2SO4, and the crude product was purified by silica gel column chromatography. The product was eluted with dichloromethane and petroleum ether to obtain a red solid, namely compound h1 (800 mg, 61.50%).

[0078] (5) Under N2 atmosphere, compound h1 (1.89 mmol) and excess iron powder (18.9 mmol) were added to a round-bottom flask containing acetic acid (30 mL), heated at 80 °C and stirred vigorously for 4 h. During this period, a large amount of solid precipitated in the system. After the reactants were consumed by thin-layer chromatography, the mixture was cooled to room temperature and an appropriate amount of distilled water was added. The crude product was filtered through diatomaceous earth, redissolved in dichloromethane, and washed with water. The organic layer was dried with anhydrous Na2SO4, the solvent was removed under reduced pressure, and the mixture was recrystallized from methanol / dichloromethane to obtain compound j1 (2.0 g, 87.46%).

[0079] (6) A mixed solution of CHCl3 (15 mL) and AcOH (5 mL) was added to a mixture of compound j1 (0.45 mmol) and compound k (0.23 mmol). The mixture was heated to 80 °C under stirring and continued for 24 h. After cooling to room temperature, the reaction mixture was poured into water and extracted three times with dichloromethane. The combined organic layers were dried with anhydrous Na2SO4 and concentrated by air separation. The crude product was purified by silica gel column chromatography with petroleum ether and dichloromethane as eluents to obtain the near-infrared II region aggregation-induced emission probe, namely compound 5BT-PBPT (400 mg, 67.43%).

[0080] The proton and carbon NMR spectra of compound 5BT-PBPT in deuterated chloroform are shown below.Figure 2 and Figure 3 As shown (the horizontal axis represents chemical shift, the same below); the mass spectrum is as follows. Figure 4 As shown.

[0081] Example 2 Following the above synthetic route, the preparation method of the near-infrared II aggregation-induced emission probe, namely compound 6BT-PBPT, includes the following steps: (1) Same as step (1) in Example 1.

[0082] (2) The only difference from step (2) in Example 1 is that compound d1 is replaced with compound d2, and finally a colorless solid, namely compound e2 (7.0 g, 83.83%), is obtained.

[0083] (3) The only difference from step (3) in Example 1 is that compound e1 is replaced with compound e2, and a gray solid, namely compound f2 (3.1 g, 71.19%), is finally obtained.

[0084] (4) The only difference from step (4) in Example 1 is that compound f1 is replaced with compound f2, and a red solid, namely compound h2 (750 mg, 57.85%), is finally obtained.

[0085] (5) The only difference from step (5) in Example 1 is that compound h1 is replaced with compound h2 to obtain compound j2 (1.8 g, 78.72%).

[0086] (6) The only difference from step (5) in Example 1 is that compound j1 is replaced with compound j2, and the near-infrared II region aggregation-induced emission probe, namely compound 6BT-PBPT (350 mg, 59.0%), is finally obtained.

[0087] The proton and carbon NMR spectra of compound 6BT-PBPT in deuterated chloroform are shown below. Figure 5 and Figure 6 As shown; mass spectrum as shown Figure 7 As shown.

[0088] test: (1) Compound 5BT-PBPT in Example 1 and compound 6BT-PBPT in Example 2 in tetrahydrofuran (THF) (10 -5 The absorption spectrum of M) is as follows: Figure 8 As shown.

[0089] The aggregation-induced emission curves of compound 5BT-PBPT from Example 1 and compound 6BT-PBPT from Example 2 in mixed solutions of tetrahydrofuran and water (with water volume fractions of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%, respectively) are shown below. Figure 9 As shown, where I This indicates the luminescence intensity in a mixed solution with a certain water content. I 0 represents the luminescence intensity in pure tetrahydrofuran. The test results show that in the mixed solution, the fluorescence of compound 5BT-PBPT in Example 1 and compound 6BT-PBPT in Example 2 increases significantly with the increase of water content. Both compounds have obvious aggregation-induced emission properties and are probes for aggregation-induced emission characteristics.

[0090] Example 3 1 mg of compound 5BT-PBPT from Example 1, 1 mg of 6BT-PBPT from Example 2, and 5 mg of DSPE-mPEG were respectively added. 2000 The solution was dissolved in 1 mL of tetrahydrofuran, and the resulting mixture was then added to 9 mL of ultrapure H₂O and immediately sonicated for 2 minutes using an ultrasonic probe at 45% output power. Subsequently, the mixture was added to a dialysis bag (molecular weight cutoff of 3500 Da) and dialyzed with deionized water in a 5 L beaker for 24 h. To remove all tetrahydrofuran, the water was replaced multiple times with fresh ultrapure water during dialysis. Finally, the nanoparticles obtained by ultrafiltration concentration were designated as 5BT-PBPT NPs and 6BT-PBPT NPs, respectively.

[0091] test: (1) The photoluminescence spectra of 5BT-PBPT NPs (final concentration 10 μM) and 6BT-PBPT NPs (final concentration 10 μM) in water in Example 3 are as follows: Figure 10 As shown, the maximum emission peaks of both are close to or exceed 1000 nm, which are typical near-infrared II luminescent materials, and they have large Stokes shift values.

[0092] (2) The photothermal conversion efficiency results of 5BT-PBPT NPs (final concentration of 50 μM) and 6BT-PBPT NPs (final concentration of 50 μM) in water under 808 nm laser irradiation are shown in the figures below. Figure 11 and Figure 12 As shown, θ represents the temperature change (ΔT) and the maximum temperature change (ΔT). maxThe ratio of the photothermal conversion efficiency (η) of 5BT-PBPT NPs to that of 6BT-PBPT NPs is 62.0%, while that of 6BT-PBPT NPs is slightly higher at 63.6%, indicating that both probes have excellent photothermal conversion capabilities.

[0093] (3) The ROS generation capability of the 5BT-PBPT NPs and 6BT-PBPT NPs in Example 3 under 808 nm laser irradiation was tested. The specific steps are as follows: 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was used as a ROS indicator to detect the ROS of an 808 nm laser (0.8 W·cm⁻¹). -2 The ROS generation of 5BT-PBPT NPs and 6BT-PBPT NPs in solution under irradiation was investigated. First, an ethanol solution of DCFH-DA (1 mM, 0.5 mL) was added to 2 mL of NaOH solution (0.01 M), and stirred at room temperature for 30 minutes to hydrolyze DCFH-DA to DCFH (dichlorodihydrodiacetic acid fluorescein). The hydrolysis product was then neutralized with 10 mL of 1×PBS (pH 7.4) to obtain an activated ROS indicator (40 μM, 12.5 mL), which was stored at 4 °C protected from light for later use. Then, the activated ROS indicator (40 μM) was mixed with 5BT-PBPT NPs or 6BT-PBPT NPs in PBS, so that the final concentration of both the ROS indicator and the 5BT-PBPT NPs (or 6BT-PBPT NPs) was 1 μM. After irradiation with an 808 nm laser for different times, the fluorescence intensity of DCFH triggered by ROS generated by 5BT-PBPT NPs or 6BT-PBPT NPs was detected using a fluorescence spectrometer to reflect the ROS generation. The excitation wavelength was 488 nm, and fluorescence signals in the range of 490-600 nm were collected.

[0094] The results are as follows Figure 13 As shown (where, I This represents the fluorescence intensity of DCFH triggered by ROS generated by 5BT-PBPT NPs. I (0 represents the fluorescence intensity of DCFH without illumination). The fluorescence intensity of DCFH of 5BT-PBPT NPs increased by nearly 1000 times after 10 minutes of 808 nm laser irradiation, while the fluorescence intensity of DCFH of 6BT-PBPT NPs increased by 240 times after 10 minutes of 808 nm laser irradiation. This indicates that both 5BT-PBPT NPs and 6BT-PBPT NPs have excellent ROS generation capabilities, and 5BT-PBPT NPs have stronger ROS generation capabilities.

[0095] (4) The dark toxicity and photothermal killing effects of 5BT-PBPT NPs (concentrations of 0 μM, 5 μM, 10 μM, 20 μM, 50 μM, 75 μM and 100 μM, respectively) on 4T1 cells were tested in Example 3. The specific steps are as follows: 4T1 cells were seeded in 96-well plates at an initial density of 5 × 10⁶ cells. 3 Cells were incubated at 100 cells / well for 24 hours. Then, cells were treated with different concentrations of 5BT-PBPT NPs. After 8 hours of incubation, cells were directly exposed to an 808 nm laser (0.8 W·cm⁻¹). -2 Cells were irradiated for 10 minutes. Cells without laser irradiation were used for dark toxicity assays. Cells were then cultured for an additional 16 hours before adding CCK-8. Finally, cell viability was recorded using a microplate reader at a wavelength of 450 nm.

[0096] The results are as follows Figure 14 As shown ( (P < 0.001) Results are expressed as the percentage of cell survival after each treatment relative to untreated control cells. 5BT-PBPT NPs exhibited low dark cytotoxicity in the 100 μM range and demonstrated excellent photothermal killing ability of 4T1 cells.

[0097] (5) The synergistic imaging (fluorescence / photoacoustic / photothermal, NIR-IIFLI / PAI / PTI) experiment of 5BT-PBPT NPs for breast cancer in Example 3 specifically includes the following steps: 4T1 tumor-bearing mice were injected with 5BT-PBPT NPs (200 μL, 2 mM). Fluorescence / photoacoustic imaging was performed at 0 h, 2 h, 6 h, 24 h, 36 h, and 48 h to detect the fluorescence / photoacoustic signals of the mouse tumors. The results are as follows: Figure 15 As shown, 5BT-PBPT NPs showed an enrichment trend in mouse tumor sites, reaching a peak at 36 h.

[0098] After fluorescence / photoacoustic imaging of 4T1 tumor-bearing mice, thermal imaging of the tumor site was performed 36 hours after injection of 5BT-PBPT NPs (200 μL, 2 mM). The results are as follows: Figure 15 As shown, the temperature of the tumor site in mice gradually increased under 808 nm laser irradiation.

[0099] Therefore, 5BT-PBPT NPs have excellent specific imaging and therapeutic capabilities for tumors.

[0100] (6) The synergistic photothermal / photodynamic therapy experiment of 5BT-PBPT NPs for breast cancer in Example 3 specifically includes the following steps: 4T1 tumor-bearing mice were randomly divided into 4 groups (n=5 in each group) and treated with different methods. The groups were named “PBS”, “PBS + L”, “5BT-PBPT NPs”, and “5BT-PBPT NPs + L”.

[0101] For the "PBS+L" group, mice were first injected with PBS (200 μL) via the tail vein, and then exposed to an 808 nm laser (0.8 W·cm²) 36 h after injection (day 0). -2 Irradiate continuously for 10 minutes; For the "PBS" group, mice were injected with PBS (200 μL) via the tail vein and were not exposed to light, serving as the control group for the "PBS+L" group; For the "5BT-PBPT NPs + L" group, mice were injected with 5BT-PBPT NPs (200 μL, 2 mM) via the tail vein and exposed to an 808 nm laser (0.8 W·cm²) 36 h post-injection (day 0). -2 Irradiate continuously for 10 minutes; For the “5BT-PBPT NPs” group, mice were injected with 5BT-PBPT NPs (200 μL, 2 mM) via the tail vein and were not exposed to light, serving as the control group for the “5BT-PBPT NPs + L” group. Tumor volume in mice was recorded every two days during treatment and calculated as V = a × b 2 / 2. (a: tumor length; b: tumor width). Relative tumor volume (RTV) is calculated as RTV = V / V0, where V0 is the initial tumor volume.

[0102] The results are as follows Figure 16 As shown, 5BT-PBPT NPs can effectively treat breast cancer tumors with photothermal / photodynamic therapy. The tumors in the "5BT-PBPT NPs + L" group completely disappeared after the second day.

[0103] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A near-infrared two-region aggregation-induced emission probe, characterized in that, The structure of the near-infrared two-region aggregation-induced emission probe is as follows: ； X1, X2 and Y are each independently selected from one of O, S, Se and Te; R1, R2 and R3 are each independently selected from one of H, F, a cyano group, a trifluoromethyl group and a saturated alkyl group. 2.The near-infrared two-region aggregation-induced emission probe of claim 1, wherein, The saturated alkyl group includes one of a methyl group, a tert-butyl group and an octyl group.

3. A method for preparing the near-infrared two-region aggregation-induced emission probe according to any one of claims 1-2, characterized in that, The preparation method of the compound shown in formula II includes the following steps: After reacting with and , the near-infrared two-region aggregation-induced emission probe is obtained.

4. The production method according to claim 3, characterized by, The preparation method of the compound shown in formula I includes the following steps: After the reaction of , hydrated ruthenium trichloride and sodium periodate, a compound shown in formula II is obtained; and / or, The preparation method of the compound shown in formula IV includes the following steps: The compound represented by formula I is obtained after mixing iron powder and acetic acid and carrying out a reaction. , iron powder and acetic acid, and carrying out a reaction.

5. The preparation method according to claim 4, characterized in that, The preparation method of the compound shown in formula V includes the following steps: After reacting with and , a compound represented by formula IV is obtained.

6. The preparation method according to claim 5, characterized in that, The preparation method of the compound shown in formula VI includes the following steps: After reacting with and , the following compounds are obtained ; The compound of formula V is obtained after reaction with with N - bromosuccinimide.

7. The preparation method according to claim 5, characterized in that, The nanoparticles include the near-infrared two-region aggregation-induced emission probe and an amphiphilic carrier coated on the surface of the near-infrared two-region aggregation-induced emission probe; the near-infrared two-region aggregation-induced emission probe is the near-infrared two-region aggregation-induced emission probe according to any one of claims 1-2 or the near-infrared two-region aggregation-induced emission probe prepared by the preparation method according to any one of claims 3-7. After reacting with and , the following compounds are obtained ; Will The mixture of 4,4'-di-tert-butyl-2,2'-bipyridine, [Ir(COD)(OMe)]2 and 4,4,5,5-tetramethyl-1,3,2-dioxoborane was reacted to give the compound shown in Formula VI.

8. A nanoparticle, characterized in that, The preparation method of the compound shown in formula II includes the following steps:

9. A method of preparing the nanoparticle of claim 8, wherein, The near-infrared two-region aggregation-induced emission probe and the amphiphilic carrier are added into water, and then the nanoparticles are obtained by sequentially performing ultrasonic treatment, dialysis and ultrafiltration concentration.

10. Use of the near-infrared two-region aggregation-induced emission probe according to any one of claims 1-2, the near-infrared two-region aggregation-induced emission probe prepared by the preparation method according to any one of claims 3-7, the nanoparticles according to claim 8 or the nanoparticles prepared by the preparation method according to claim 9 in the preparation of a multimodal diagnosis and treatment integrated nanodrug. ​

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