Near-infrared two-region fluorescent molecule and preparation method and application thereof

Abstract

The application discloses a near-infrared two-region fluorescent molecule and a preparation method and application thereof. The fluorescent molecule is prepared by conjugating a non-planar tricyclic structure phenothiazine group with strong electron supply capacity and active oxygen generation capacity with a strong electron acceptor benzo-bisthiazole core, and further introducing a tetraphenyl ethylene module to endow AIE characteristics. The fluorescent molecule exhibits NIR-II fluorescent emission at 1083 nm, the fluorescent quantum yield is 1.53%, the photo-thermal conversion efficiency is as high as 63%, and the fluorescent molecule has I-type ROS generation capacity, so that the fluorescent molecule can be used as a near-infrared two-region fluorescent imaging contrast agent to realize multi-modal imaging of living blood vessels and tumor sites and photo-thermal-photo-dynamic synergistic treatment guided by imaging.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and in particular relates to a near-infrared second-region fluorescent molecule and a preparation method and application thereof. Background Art

[0002] Traditional cancer treatments, including surgery, chemotherapy, and radiotherapy, have shown good therapeutic effects in some cases. However, the above traditional treatments are often accompanied by high recurrence rates, significant side effects, and invasive procedures. Currently, near-infrared II (NIR-II) organic small molecules are considered to be promising candidate molecules in tumor imaging and diagnosis due to their excellent properties. These properties include easy structural modification, tunable optical properties, and good biocompatibility. However, the application of fluorescent organic small molecules is generally limited to single-modality imaging. At the same time, multifunctional organic small molecules that can combine multimodal imaging (such as fluorescence imaging, photoacoustic imaging, and photothermal imaging) with therapeutic methods (such as photothermal therapy, photodynamic therapy, and gas therapy) are being widely studied.

[0003] However, the practical application of traditional fluorescent organic small molecules (such as indocyanine green and methylene blue) is often limited by the following defects: limited fluorescence penetration depth, low photostability, and aggregation-induced quenching (ACQ) effects caused by strong π-π interactions in large conjugated systems. These defects significantly restrict their application in tumor diagnosis and treatment. In contrast, organic small molecules with aggregation-induced emission (AIE) properties have shown significant advantages in multimodal tumor imaging and photothermal-photodynamic synergistic therapy. AIE materials exhibit strong luminescence properties in the aggregated state, which can significantly improve the signal-to-noise ratio and sensitivity of imaging. In addition, the multi-rotational structure of AIE molecules achieves a controllable balance between radiative energy and non-radiative energy relaxation pathways in the aggregated state, making multifunctional diagnostic and therapeutic platforms based on single AIE molecules possible. In addition, AIE molecules are of great research significance due to their large Stokes shift, excellent photostability, and ability to passively target tumors. Currently, a variety of NIR-II AIE molecules have been successfully used for multimodal imaging and therapy. However, the design and synthesis of AIE molecules that achieve high fluorescence quantum yield, high photothermal conversion efficiency, and robust reactive oxygen species generation capabilities still face key challenges. Addressing these challenges is particularly important for promoting the practical application of AIE molecules in cancer diagnosis and treatment. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a near-infrared second region fluorescent molecule having aggregation-induced emission (AIE) properties, which can achieve efficient multimodal imaging.

[0005] The present invention also provides a method for preparing the aforementioned near-infrared second-region fluorescent molecules.

[0006] The present application also provides a composition.

[0007] The present application also provides a preparation method of the composition.

[0008] The present application also provides an application of the near-infrared two-region fluorescent molecule.

[0009] The present application also provides an application of the composition.

[0010] According to a first aspect of the present application, a near-infrared two-region fluorescent molecule is provided, and the chemical structural formula of the near-infrared two-region fluorescent molecule is as follows:

[0011]

[0012] wherein R is selected from any one of an alkyl group, a hydroxyl-C 1-6 an alkyl group, a substituted or unsubstituted phenyl group;

[0013] The substituent group of the substituted or unsubstituted phenyl group includes a hydroxyl group.

[0014] In some embodiments of the present application, the R is a hydroxypropyl group.

[0015] According to a second aspect of the present application, a preparation method of a near-infrared two-region fluorescent molecule is provided, and the preparation method comprises the following steps:

[0016] S1: sodium hydride, phenothiazine and 3-bromopropanol are added into N,N-dimethylformamide to perform a substitution reaction, and an intermediate 1 shown in formula II is obtained through column chromatography purification;

[0017]

[0018] S2: the intermediate 1, N-bromosuccinimide and tetrahydrofuran are uniformly mixed to perform a substitution reaction, and an intermediate 2 shown in formula III is obtained through column chromatography purification;

[0019]

[0020] S3: the intermediate 2, bis(pyrrolidinato)diboron, potassium acetate, 1,4-dioxane and a palladium catalyst are uniformly mixed to perform a Suzuki coupling reaction, and an intermediate 3 shown in formula IV is obtained through column chromatography purification;

[0021]

[0022] S4: Intermediate 3, (2-bromoethylene-1,1,2-triyl)triphenyl, 4,8-bis(5-bromo-3-(2-ethylhexyl)-2-thienyl)-2λ4δ2-benzo[1,2-c:4,5-c']bis[1,2,5]thiadiazole, palladium catalyst and potassium carbonate are mixed and subjected to Suzuki coupling reaction, and the mixture is purified by column chromatography to obtain the near-infrared second-region fluorescent molecule BPT as shown in Formula V;

[0023]

[0024] In some embodiments of the present invention, the molar ratio of sodium hydride, phenothiazine and 3-bromopropanol in step S1 is (1-4):1:1.

[0025] In some embodiments of the present invention, the molar ratio of the intermediate 1 to N-bromosuccinimide in step S2 is 1:(1-4), and the substitution reaction time is 16-32 hours.

[0026] In some embodiments of the present invention, the molar ratio of the intermediate 2, bis(pyrrolidino)diboron, potassium acetate and palladium catalyst in step S3 is 1:(2-4):(4-8):(0.5-1), and the molar volume ratio of the intermediate 2 to 1,4-dioxane is 1:(2-4) mmol / mL.

[0027] In some embodiments of the present invention, the reaction temperature of the Suzuki coupling reaction in step S3 is 70-90° C., and the reaction time is 30-42 h.

[0028] In some embodiments of the present invention, the molar ratio of the intermediate 3, (2-bromoethylene-1,1,2-triyl)triphenyl, 4,8-bis(5-bromo-3-(2-ethylhexyl)-2-thienyl)-2λ4δ2-benzo[1,2-c:4,5-c']bis[1,2,5]thiadiazole, palladium catalyst and potassium carbonate in step S4 is (20-30):1:(6-8):1:(30-40).

[0029] In some embodiments of the present invention, the reaction temperature of the Suzuki coupling reaction in step S4 is 60-80° C., and the reaction time is 16-32 h.

[0030] In some embodiments of the present invention, the palladium catalyst is selected from at least one of tetrakis(triphenylphosphine)palladium, palladium dichloride, palladium acetate, and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II).

[0031] According to a third aspect of the present invention, a composition is provided, comprising the near-infrared second region fluorescent molecule, a nitric oxide prodrug, and an amphiphilic polymer carrier as described in the first aspect of the present invention;

[0032] The mass ratio of the near-infrared second-region fluorescent molecule, the nitric oxide prodrug and the amphiphilic polymer carrier is (0.5-2): (0.02-0.1):10.

[0033] In some embodiments of the present invention, the mass ratio of the near-infrared second region fluorescent molecule, the nitric oxide prodrug and the amphiphilic polymer carrier is (0.5-1.5): (0.02-0.08):10.

[0034] In some embodiments of the present invention, the mass ratio of the near-infrared second region fluorescent molecule, the nitric oxide prodrug and the amphiphilic polymer carrier is (0.8-1.2): (0.04-0.06):10.

[0035] In some embodiments of the present invention, the nitric oxide prodrug includes at least one of an organic nitrate compound, a nitroprusside compound, an S-nitrosothiol compound, a furan nitrogen oxide compound, a hydroxamic acid compound, and an azodiol olefinium salt compound.

[0036] In some embodiments of the present invention, the nitric oxide prodrug is an azodiol alkene salt compound.

[0037] In some embodiments of the present invention, the azodicarbonium salt compound is O2-(2,4-dinitrophenyl)-1-[(4-ethoxycarbonyl)piperazinyl]diazenium-1,2-diol ester (JS-K).

[0038] In some embodiments of the present invention, the amphiphilic polymer carrier includes at least one of distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-mPEG2000), polylactic acid-co-glycolic acid (PLGA) and polyoxyethylene polyoxypropylene ether triblock copolymer (Pluronic F-127).

[0039] According to a fourth aspect of the present invention, a method for preparing the composition according to the third aspect of the present invention is provided, the preparation method comprising the following steps:

[0040] The near-infrared second-zone fluorescent molecule, nitric oxide prodrug, and amphiphilic polymer carrier are dissolved in an organic solvent to obtain a mixed solution; the mixed solution is added to water under ultrasonic treatment for self-assembly, and the organic solvent is removed to obtain a composition.

[0041] In some embodiments of the present invention, the organic solvent comprises tetrahydrofuran (THF).

[0042] In some embodiments of the present invention, the volume ratio of the mixed liquid to water is 1:(7-12).

[0043] In some embodiments of the present invention, the volume ratio of the mixed liquid to water is 1:(9-11).

[0044] In some embodiments of the present invention, the organic solvent is removed by nitrogen bubbling.

[0045] According to the fifth aspect of the present invention, the use of the near-infrared second region fluorescent molecule as described in the first aspect of the present invention or the composition as described in the third aspect of the present invention in the preparation of a fluorescent probe is proposed.

[0046] In some embodiments of the present invention, the fluorescent probe comprises a fluorescent probe for at least one of (1)-(3):

[0047] (1) Tumor imaging;

[0048] (2) Cardiovascular imaging;

[0049] (3)Neuroimaging.

[0050] According to a sixth aspect of the present invention, a use of the composition according to the third aspect of the present invention in the preparation of a product for diagnosing and / or treating tumors is proposed.

[0051] In some embodiments of the present invention, the product for diagnosing and / or treating tumors comprises a photodynamic nano-drug delivery system. In some embodiments of the present invention, the tumor comprises a solid tumor.

[0052] The present invention has at least the following beneficial effects:

[0053] The present invention provides a near-infrared zone II fluorescent molecule, which is conjugated by a non-planar tricyclic phenothiazine group with strong electron donation and reactive oxygen species (ROS) generation capabilities with a strong electron acceptor benzobithiadiazole (BBTD) core, and further introduced a tetraphenylethylene (TPE) module to impart AIE properties; the fluorescent molecule exhibits NIR-II fluorescence emission at 1083nm, a fluorescence quantum yield of 1.53%, a photothermal conversion efficiency of up to 63%, and has type I ROS generation capabilities; BPT can be used as a near-infrared zone II fluorescent imaging contrast agent to achieve multimodal imaging of living blood vessels and tumor sites, as well as imaging-guided photothermal-photodynamic synergistic therapy.

[0054] The present invention also provides a composition containing the aforementioned near-infrared second region fluorescent molecule, wherein the composition co-encapsulates the aforementioned fluorescent molecule and the nitric oxide prodrug JS-K; JS-K is a tumor-suppressing drug that can efficiently consume reduced glutathione (GSH) in tumor cells and release NO. The NO released by JSK reacts with superoxide radicals (O 2-) efficiently bind in the tumor microenvironment, form peroxynitrite (ONOO - ), thereby further inducing apoptosis of cancer cells; under 808 nm laser (0.7 W / cm 2 ) irradiation, the composition exhibits significant cytotoxicity to tumor cells at a low concentration. The composition achieves high-quality multi-modal imaging of blood vessels and tumor regions in vivo, while exhibiting superior photodynamic therapy effect, achieving complete ablation of tumors with only one treatment.

[0055] Additional aspects and advantages of the present application will be partially given in the following description and partially will become apparent from the following description. BRIEF DESCRIPTION OF DRAWINGS

[0056] The present application will be further described below in conjunction with the accompanying drawings and examples, in which:

[0057] Figure 1 NMR hydrogen spectrum of compound 1c prepared for example 1 of the present application;

[0058] Figure 2 NMR carbon spectrum of compound 1c prepared for example 1 of the present application;

[0059] Figure 3 NMR hydrogen spectrum of compound 2b prepared for example 1 of the present application;

[0060] Figure 4 NMR carbon spectrum of compound 2b prepared for example 1 of the present application;

[0061] Figure 5 NMR hydrogen spectrum of compound 3c prepared for example 1 of the present application;

[0062] Figure 6 NMR carbon spectrum of compound 3c prepared for example 1 of the present application;

[0063] Figure 7 Fluorescence imaging results of blood vessels in mice of BPT NP in the test example of the present application;

[0064] Figure 8 NIR-II excitation fluorescence, photoacoustic and photothermal tumor imaging results of BPT NP in tumor-bearing mice in the test example of the present application, wherein A and B are NIR-II excitation fluorescence, photoacoustic imaging results at 4, 12, 24 and 48 hours after administration, respectively, and C is the infrared photothermal imaging result after 12 hours of administration;

[0065] Figure 9 Detection results of reactive oxygen species of 4T1 cells treated by JS-K-BPT NP in the test example of the present application;

[0066] Figure 10 The figure shows the results of reactive oxygen species detection in 4T1 cells after treatment with JS-K-BPT NPs in the experimental example of the present invention, where the scale bar is 30 μm;

[0067] Figure 11 The cell viability results of 4T1 cells treated under different conditions in the experimental examples of the present invention are shown in Figures A to D, respectively, corresponding to the cell viability of groups (1) to (4);

[0068] Figure 12 Figures 2 and 3 are the results of live and dead cell detection under a microscope of 4T1 cells treated under different conditions in the experimental examples of the present invention, wherein the scale bar in Figure A is 100 μm;

[0069] Figure 13 This is a flow cytometry result diagram of apoptosis detection of 4T1 cells treated under different conditions in the experimental examples of the present invention;

[0070] Figure 14 This is a curve diagram of tumor volume changes in tumor-bearing mice under different treatment conditions in the experimental examples of the present invention;

[0071] Figure 15 The figure shows the quantum yield measurement results of BPT NPs in the experimental examples of the present invention;

[0072] Figure 16 Graph showing the linear fitting results of the cooling period of BPT NPs and the negative natural logarithm of the driving force temperature in the experimental example of the present invention. DETAILED DESCRIPTION

[0073] The following describes embodiments of the present invention in detail. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended only to explain the present invention and are not to be construed as limiting the present invention.

[0074] In the description of the present invention, reference to terms such as "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the exemplary expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0075] Example 1 Synthesis of Near-Infrared Region II Fluorescent Molecules

[0076] This example prepares a near-infrared second-region fluorescent molecule BPT, and its synthesis path and specific steps are as follows:

[0077]

[0078] Step 1:

[0079]

[0080] NaH (600 mg, 25 mmol) was suspended in DMF (15 mL) under ice conditions, followed by the addition of Compound 1a (phenothiazine) (2.0 g, 10.0 mmol) and Compound 1b (3-bromopropanol) (10.0 mmol) to obtain a suspension. The suspension was stirred at room temperature overnight. After the reaction was complete and cooled to room temperature, a saturated aqueous sodium chloride solution was added. The organic layer was collected, extracted with dichloromethane, and dried over anhydrous NaSO. After evaporation of the organic solvent, the crude product was purified by column chromatography to obtain the desired product, Compound 1c (0.85 g, 35% yield), as a purple oil.

[0081] The NMR results of compound 1c are as follows: 1 H NMR(400MHz, DMSO-d6)δ7.24-7.06(m,4H),7.05(dd,J=8.2,1.2Hz,2H),6.94(td,J=7.4,1.1H z, 2H), 4.59 (t, J = 5.0Hz, 1H), 3.94 (t, J = 6.9Hz, 2H), 3.53 (q, J = 5.8Hz, 2H), 1.90-1.80 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 150.00, 132.79, 132.29, 128.70, 127.62, 120.98, 63.29, 48.61, 34.78. NMR spectrum is shown in Figure 1 and Figure 2 shown.

[0082] Step 2:

[0083]

[0084] Compound 1c (10 mmol), N-bromosuccinimide (NBS, 20 mmol), and 50 mL of THF were added to a flask and stirred at room temperature for 24 hours. After the reaction, silica gel was added directly, and the THF was removed by rotary evaporation. The crude product was purified by column chromatography using petroleum ether as the eluent to obtain compound 2a.

[0085] The crude product 2a, which had been initially purified, was mixed with bis(pyrrolidino)diboron (7.62 g, 30 mmol), potassium acetate (KOAc, 5.88 g, 60 mmol), 1,4-dioxane (30 mL), and Pd(dppf)Cl2 (0.365 g, 0.5 mmol), then sealed with parafilm. The reaction system was evacuated through three freeze-vacuum-thaw cycles to remove air. The reaction system was heated to 80°C for 36 hours. After completion of the reaction, the mixture was cooled to room temperature and washed with water. The organic layer was collected and dried over anhydrous Na2SO4 to obtain the crude product. The crude product was further purified by column chromatography using a petroleum ether / dichloromethane mixture as the eluent to obtain compound 2b as a pale yellow solid in a 30% yield (1.6 g, 3 mmol).

[0086] The NMR results of compound 2b are as follows: 1 H NMR (400MHz, Chloroform-d) δ7.49 (d, J = 1.3 Hz, 4H), 6.82 (d, J = 7.8 Hz, 2H), 3.99 (s, 2H), 3.69 (t, J = 5.9 Hz, 2H), 1.95 (t, J = 6.2 Hz, 2H), 1.25 (s, 24H). 13 C NMR (101 MHz, Chloroform-d) δ 147.33, 134.11, 133.93, 124.59, 114.99, 83.75, 60.34, 44.04, 29.38, 24.83. The mass spectrum data of compound 2b are as follows: HRMS: (ESI): calcd.for [C 27 H 38 O5NB2S,M+H] + ,510.26513;found,510.26568. NMR spectrum is as follows Figure 3 and Figure 4 shown.

[0087] Step 3:

[0088]

[0089] A mixture of 4,8-bis(5-bromo-3-(2-ethylhexyl)-2-thienyl)-2λ4δ2-benzo[l,2-c:4,5- c']bis[l,2,5]thiadiazole (compound 3b, 500 mg, 0.73 mmol), 3-(3,7-bis(4,4,5,5-tetramethyl- 1,3,2-dioxaborolan-2-yl)-10H-phenothiazin-10-yl)propan-l-ol (compound 2b, 837 mg, 1.83 mmol), (2-bromovinyl-l,l,2-triyl)triphenyl (compound 3a, 84 mg, 0.073 mmol), tetrakis(triphenylphosphine)palladium (Pd(PPh3)4, 84 mg, 0.073 mmol), and potassium carbonate (K2CO3, 353 mg, 2.56 mmol) were added into a mixture of tetrahydrofuran / water (5:1, 60 mL) and heated at 70 °C under nitrogen atmosphere for 24 h. After the reaction was completed, the reaction mixture was cooled to room temperature, extracted with dichloromethane (DCM) three times, and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The resulting concentrate was purified by silica gel column chromatography using dichloromethane and n-hexane as eluents to obtain the target product, compound 3c (510 mg, 9% yield), a near-infrared two-region fluorescent molecule, BPT.

[0090] The NMR results of BPT are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 7.72 - 7.33 (m, 8H), 7.29 - 6.48 (m, 36H), 4.60 (s, 2H), 3.87 (s, 4H), 3.51 (d, J = 4.9 Hz, 4H), 1.80 (d, J = 6.9 Hz, 4H), 1.23 - 1.09 (m, 4H), 0.99 - 0.61 (m, 18H), 0.50 (d, J = 6.3 Hz, 6H), 0.34 (s, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 153.22, 144.81, 143.78, 131.28, 131.19, 131.13, 130.69, 127.81, 127.67, 127.59, 126.49, 126.31, 125.43, 124.84, 124.36, 116.17, 115.79, 115.75, 115.66, 114.57, 83.76, 60.22, 40.48, 34.33, 32.44, 28.54, 25.60, 24.64, 22.80, 13.80, 10.41. The mass spectral data of BPT are as follows: HRMS (ESI): calcd. for [C 100 H 93 O2N6S6M+H] +:calcd 1601.56788, vfound 1601.56628. NMR spectrum is shown in Figure 5 and Figure 6 shown.

[0091] Example 2 Preparation of the composition

[0092] This example prepares a composition (JS-K-BPTNP) based on the near-infrared second region fluorescent molecule BPT prepared in Example 1. The specific preparation method is as follows:

[0093] Under 180W, 40kHz ultrasonic treatment, 1 mL of THF solution containing 1 mg BPT, 50 μg JS-K (purchased from Yuanye Bio, T23314) and 10 mg DSPE-mPEG2000 (purchased from Shenzhen Meiluo Technology Co., Ltd., 020805) was poured into 9 mL of deionized water and maintained for 10 minutes. Then, THF and excess deionized water in the system were removed by nitrogen bubbling on a hot plate to obtain an aqueous solution containing the composition, in which the BPT content in the aqueous solution was 1 mg / mL.

[0094] Example 3

[0095] This example prepared a nanoparticle (BPTNP) based on the near-infrared second-region fluorescent molecule BPT prepared in Example 1. The only difference from Example 2 is that the JS-K component is omitted, and the remaining steps are consistent with Example 2.

[0096] Test example

[0097] This experimental example tested the in vivo imaging performance of the fluorescent molecule BPT provided in Example 3, and also tested the effect of the nanoparticles prepared in Examples 2 and 3 in tumor photodynamic therapy. The specific experimental methods and results are as follows:

[0098] 1. Fluorescence Imaging of Vascular Vasculature in Mice Using BPT NPs

[0099] Five-week-old female BALB / c nude mice were anesthetized with isoflurane and then intravenously injected with BPT NP solution (calculated as BPT: 2 mg / mL, 100 μL per mouse) through the tail vein. Immediately after injection, the mice were imaged using the NIR-OPTICS SeriesIII 900 / 1700 small animal imaging system for fluorescence imaging. The imaging results are shown in Figure 2. Figure 7 shown.

[0100] Depend on Figure 7 It can be seen that the near-infrared second region fluorescent molecule BPT provided by the present invention can exhibit good near-infrared second region fluorescence imaging performance in mice.

[0101] 2. NIR-II-stimulated fluorescence, photoacoustic, and photothermal tumor imaging of BPT NPs in tumor-bearing mice

[0102] For NIR-II fluorescence and photoacoustic imaging, 4T1 breast tumor-bearing mice were first constructed. All animal experiments were performed in accordance with the governmental and international guidelines for the care and use of laboratory animals from the Ministry of Science and Technology of the People's Republic of China and approved by the Animal Care and Utilization Committee of the Southern University of Science and Technology (resolution number: SUST-JY202406114). All mice were purchased from the Guangdong Medical Laboratory Animal Center (GDMLAC). For 5-week-old female BALB / c mice, 1 × 10 6 4T1 cells were subcutaneously injected into the posterior thigh to establish a 4T1 tumor model in BALB / c mice.

[0103] 4T1 breast tumor-bearing mice were injected with 100 μL of BPT NP solution (BPT at 1.6 mg / mL) via the tail vein. In vivo NIR-II fluorescence images were then captured at different time points (4, 12, 24, and 48 hours) after injection using an NIR-II in vivo imaging system with an 1100 nm long-pass (LP) filter and 808 nm excitation (fluorescence imaging was acquired using a NIR-OPTICS Series III 900 / 1700 small animal imaging system, purchased from Suzhou NIR-OPTICS Technology Co., Ltd.). The results are shown in Figure 3. Figure 8 As shown in Figure A. In addition, after intravenous injection of BPT NPs, photoacoustic imaging was performed at specified time intervals on a photoacoustic imaging system. The photoacoustic imaging system was constructed based on a US commercial platform (Vantage-128-HF, Verasonics, USA). A PA / US dual-mode imaging system was constructed, using a tunable laser (Surelite OPO, Continuum, CA) as the excitation light source and a linear transducer (L11-5v, Verasonic, USA) to emit ultrasound and detect PA signals and acoustic echoes. The laser pulse duration was 7 ns, the laser repetition rate was 20 Hz, and the center frequency of the linear transducer was 7.5 MHz. The results are shown in Figure 4. Figure 8 As shown in B.

[0104] For in vivo photothermal imaging, 12 hours after the tumor-bearing mice were injected with BPT NP solution, the images were taken with an infrared camera under the conditions of 808 nm laser (0.7 W / cm 2 ) Infrared thermal images of mice were collected under different irradiation times, and the results were as follows Figure 8 C. Mice injected with normal saline under the same irradiation conditions were used as controls.

[0105] Depend on Figure 8It can be seen that the near-infrared zone II fluorescent molecule BPT provided by the present invention has good tumor targeting, can clearly image tumors in tumor-bearing mice, and is suitable for wide application in clinical diagnosis and treatment methods such as near-infrared zone II fluorescence imaging and photothermal imaging.

[0106] 3. In vitro detection of the effect of JS-K-BPT NP on the generation of reactive oxygen species in 4T1 cells

[0107] In this experiment, commercially available DHR123, DAF-FM DA and O56 probes were used to detect O2 - ,NO and ONOO - The control groups of the following experiments were all treated with 1 μM JS-K (1% DMSO was added to assist in dissolution), and the images were taken under 488 nm excitation light, and the fluorescence of the corresponding probe was measured at 525 nm in a PL instrument. The 4T1 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences.

[0108] 1) O2 - A mixture of probe DHR123 (10 μM) and JS-K-BPT NP prepared in Example 2 (wherein the BPT content was 5 μM and the JS-K content was 1 μM) was added to the culture system of 4T1 cells and the cells were cultured with or without 808 nm laser irradiation (0.7 W / cm 2 ) was taken and the fluorescence intensity was recorded. The results were as follows Figure 9 A and Figure 10 shown.

[0109] 2) To detect NO, 5 mL of DAF-FM DA ethanol solution (1×10 -3 mol / L) was added with 2 mL of NaOH (1×10 -2 mol / L) for 30 minutes. At this time, DAF-FM DA was hydrolyzed into DAF-FM, and then a mixture of the hydrolyzed DAF-FM (10 μM), JS-K-BPT NPs prepared in Example 2 (wherein the BPT content was 5 μM and the JS-K content was 1 μM) and glutathione (1 mM) was added to the culture system of 4T1 cells, and the cells were cultured with or without 808 nm laser irradiation (0.7 W / cm 2 ) was taken and the fluorescence intensity was recorded. The results were as follows Figure 9 B and Figure 10 shown.

[0110] 3) ONOO -A mixture of probe O56 (10 μM) and JS-K-BPT NPs prepared in Example 2 (wherein the BPT content was 5 μM and the JS-K content was 1 μM) was added to the culture system of 4T1 cells and the cells were cultured under conditions with or without 808 nm laser irradiation (0.7 W / cm 2 ) was taken and the fluorescence intensity was recorded. The results were as follows Figure 9 C and Figure 10 shown.

[0111] Depend on Figure 9 It can be seen that the JS-K-BPT NP provided in Example 2 can only produce NO through JS-K when there is no 808nm laser irradiation, but after irradiation with 808nm laser, BPT plays its role in generating type I reactive oxygen species and produces O2 - , O2 - Further reacts with NO to form ONOO - , thereby enhancing the ability to kill tumors.

[0112] 4. In vitro detection of the effect of JS-K-BPT NP on the cell viability of 4T1 cells

[0113] 1) CCK-8 method:

[0114] 4T1 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin-streptomycin) at 37°C in a humidified atmosphere containing 5% carbon dioxide. 4T1 cells were plated at 5 × 10 cells per well. 3 The cells were plated on a 96-well plate at a density of 100 cells / well and incubated for 24 hours. Subsequently, the cells were divided into four groups (I)-(IV) for testing:

[0115] (I) After the growth medium was removed, it was replaced with fresh RPMI1640 medium containing different concentrations of BPT NPs (0, 2, 5, 10, 20, and 50 μM) and incubated in the dark for 10 h;

[0116] (II) After the growth medium was removed, it was replaced with fresh RPMI1640 medium containing different concentrations of BPT NPs (0, 2, 5, 10, 20, and 50 μM). After incubation in the dark for 6 h, the cells were irradiated with 808 nm laser (0.7 W / cm 2 ) for 10 minutes, and then continue incubation in the dark for 4 hours;

[0117] (III) After the growth medium was removed, it was replaced with fresh RPMI1640 medium containing different concentrations of JS-K (0, 0.4, 0.8, 2, 4, and 10 μM) and incubated in the dark for 10 h;

[0118] (IV) After the growth medium was removed, it was replaced with fresh RPMI1640 medium containing different concentrations of JS-K-BPT NPs (BPT at 0, 2, 5, 10, 20, and 50 μM, JS-K at 0, 0.4, 0.8, 2, 4, and 10 μM). After incubation for 6 h in the dark, the cells were irradiated with 808 nm laser light (0.7 W / cm 2 ) for 10 minutes, and then continue incubation in the dark for 4 hours;

[0119] After the incubation period for groups (I)-(IV) above, the culture medium was discarded and the cells were washed three times with PBS. Subsequently, the cells were incubated in fresh FBS-free RPMI1640 culture medium supplemented with 10% CCK-8 in the dark for 1 hour. Finally, the absorbance of the samples was quantified at a wavelength of 450 nm using a microplate reader. The results were expressed as the percentage of viable cells after different treatments compared to untreated control cells. The relative cell viability was determined using the following formula: Cell viability (%) = (OD sample - OD background) / (OD control - OD background) × 100%. The results are shown in Table 1. Figure 11 shown.

[0120] Depend on Figure 11 It can be seen that when cells are treated with BPT NP without laser treatment, cell viability can only be reduced when treated with high concentration (50 μM). After laser treatment, only 10 μM BPT NP is required to achieve the effect of 50 μM without laser treatment. Under the conditions of laser and 50 μM BPT NP, the survival rate of 4T1 cells dropped to below 20%; when cells were treated with JS-K alone, cell activity could only be inhibited to a certain extent when treated with high concentration; after administering JS-K-BPT NP prepared in Example 2 of the present invention and laser treatment, the survival rate of tumor cells was greatly reduced, and the inhibitory effect was far better than that of JS-K or BPT NP alone.

[0121] 2) Calcein AM / PI method for live and dead cell staining:

[0122] 4T1 cells were plated at 2 × 10 4 The cells were plated on confocal culture dishes at a density of 100 cells / mL and incubated for 24 hours. Subsequently, 4T1 cells were exposed to different treatments:

[0123] (a) Incubate with PBS for 6 h;

[0124] (b) Incubated with PBS for 6 h, followed by 808 nm laser irradiation for 10 min;

[0125] (c) incubated with BPT NPs (BPT concentration of 20 μM) for 6 h;

[0126] (d) incubated with JS-K (4 μM) for 6 h;

[0127] (e) Incubated with BPT NPs (BPT concentration was 20 μM) for 6 h and then irradiated with 808 nm laser for 10 min;

[0128] (f) Incubated with JS-K-BPT NPs (JS-K 4 μM, BPT 20 μM) for 6 h and then irradiated with 808 nm laser for 10 min;

[0129] The power of the above 808nm laser is 0.7W cm -2 . After the above-mentioned (a)-(f) treatments, each group of cells was further incubated at 37°C for 30 minutes. Subsequently, the cells were stained using a Calcein AM / PI double staining kit according to the manufacturer's instructions (Calcein AM / PI double staining kit was purchased from Elabscience). After staining, the cells were carefully washed and then imaged using an Olympus IX83 inverted microscope. Imaging conditions: Excitation wavelength: Calcein AM is 488 nm, PI is 561 nm; Emission filter: Calcein AM is 500-550 nm, PI is 600-700 nm. The results are shown in Figure 2. Figure 12 shown.

[0130] Depend on Figure 12 It can be seen that green represents live cells and red represents dead cells. Under the conditions of laser and JS-K-BPT NP (JS-K 4 μM, BPT 20 μM), a large number of 4T1 cells died.

[0131] 3) Cell apoptosis detection:

[0132] 4T1 cells were plated at 2 × 10 4 The cells were plated at a density of 100 cells / well in 12-well plates and incubated for 24 hours. Subsequently, 4T1 cells were exposed to different treatments:

[0133] ① Incubate with PBS for 6 hours;

[0134] ② Incubate with PBS for 6 hours, then irradiate with 808nm laser for 10 minutes;

[0135] ③ Incubate with BPT NPs (BPT concentration is 20 μM) for 6 h;

[0136] ④ Incubate with JS-K (4 μM) for 6 hours;

[0137] ⑤ Incubate with BPT NPs (BPT concentration is 20 μM) for 6 hours, and then irradiate with 808 nm laser for 10 minutes;

[0138] ⑥ Incubate with JS-K-BPT NPs (JS-K 4 μM, BPT 20 μM) for 6 h, followed by 808 nm laser irradiation for 10 min;

[0139] The power of the above 808nm laser is 0.7W cm -2 After the above treatments ①-⑥, the cells were further incubated for 0.5 hours. Afterwards, the cells were washed with PBS and then centrifuged at 1000 rpm for 5 minutes at 4°C to collect the cell pellet. The collected samples were then stained using the Annexin V-FITC / PI apoptosis detection kit according to the manufacturer's instructions and analyzed by flow cytometry. The results are shown in Figure 2. Figure 13 As shown, Figure 13 The first row of flow cytometry plots corresponds to the results of ①-③ from left to right, and the second row of flow cytometry plots corresponds to the results of ④-⑥ from left to right; among them, the Q4 quadrant represents normal cells, and the Q2 quadrant represents late apoptotic cells.

[0140] Depend on Figure 13 It was found that under the conditions of laser and JS-K-BPT NP (JS-K 4 μM, BPT 20 μM), 4T1 cells underwent apoptosis in large quantities.

[0141] 5. Anti-tumor effects of nanoparticles

[0142] To investigate the in vivo antitumor efficacy of BPT NPs and JS-K-BPT NPs, the tumors were cultured and plated when the tumor volume reached approximately 100 mm. 3 At the same time, mice bearing 4T1 breast tumors were randomly divided into five groups (4 mice in each group), named Control, BPT NPs, JSK-BPT NPs, BPT NPs+light, and JSK-BPT NPs+light.

[0143] Among them, the BPT NPs group mice were injected with 100 μL of nanoparticles BPT NPs (BPT content of 1.6 mg / mL) prepared in Example 3 through the tail vein, and the JSK-BPT NPs mice were injected with 100 μL of nanoparticles JS-K-BPT NPs (BPT content of 1.6 mg / mL, JS-K content of 0.08 mg / mL) prepared in Example 2 through the tail vein. These two groups of mice did not need to be irradiated with laser. The control group mice were given 100 μL of nanoparticles BPT NPs (BPT content of 1.6 mg / mL) prepared in Example 3 through the tail vein; the BPT NPs+light group mice were injected with 100 μL of nanoparticles BPT NPs (BPT content of 1.6 mg / mL) prepared in Example 3 through the tail vein, and 12 hours after the injection, the tumor site was irradiated with 808 nm laser (0.7 W / cm 2) for 10 minutes; mice in the JSK-BPT NPs+light group were injected with 100 μL of JS-K-BPT NPs (BPT content of 1.6 mg / mL, JS-K content of 0.08 mg / mL) prepared in Example 2 via tail vein, and 12 hours after injection, the tumor site was illuminated with 808 nm laser (0.7 W / cm 2 ) for 10 minutes.

[0144] During the 12-day experimental period, the weight and tumor volume of mice were recorded every 3 days to calculate the changes in weight and relative tumor volume. Tumor size was measured with a vernier caliper and the formula V = (length × width) was used. 2 ) / 2 to estimate the tumor volume; the results are as follows Figure 14 shown.

[0145] Depend on Figure 14 It can be seen that after laser irradiation, the tumor size of mice in the BPT NPs+light group and the JSK-BPT NPs+light group was significantly reduced compared with the control group, indicating that the BPT NPs and JS-K-BPT NPs provided by the present invention both have excellent photodynamic therapy effects, and the combined use of JS-K and BPT can further amplify the anti-tumor effect of JS-K, and the two have a synergistic effect.

[0146] 6. Quantum Yield Measurement of BPT NPs

[0147] BPT NPs were formulated into five aqueous solutions with absorbance values ​​of 0.02, 0.04, 0.06, 0.08, and 0.1, and the fluorescence intensity of each solution was measured at 808 nm. The fluorescence intensity of IR26 dichloromethane solutions with absorbance values ​​of 0.02, 0.04, 0.06, 0.08, and 0.1 was measured under the same conditions. The quantum yield was calculated using the following equation:

[0148]

[0149] Among them, QY s and QY r are the fluorescence quantum yields of the sample to be tested and the reference standard (IR26 = 0.5% dichloromethane solution), A is the absorbance, F is the relative integrated fluorescence intensity, n is the refractive index of the solvent, r and s represent the sample to be tested and the reference standard respectively; the results are shown in Figure 2. Figure 15 shown.

[0150] Depend on Figure 15 It can be seen that the fluorescence quantum yield of BPT NPs reaches 1.53%.

[0151] 7. Photothermal performance test of BPT NPs

[0152] The BPT-NP aqueous solution (50 μM calculated as BPT) was irradiated with an 808 nm laser at a designed power density, and the temperature was recorded at 20-second intervals for 8 minutes. The photothermal conversion efficiency in the aqueous solution was calculated according to the reference (D. Xi, M. Xiao, J. Cao, L. Zhao, N. Xu, S. Long, J. Fan, K. Shao, W. Sun, X. Yan, and X. Peng, Adv. Mater. 2020, 32, 1907855.). The results are shown in Figure 2. Figure 16 shown.

[0153] Depend on Figure 16 It can be seen that the photothermal conversion efficiency of BPT NPs is as high as 63%.

[0154] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A near-infrared second region fluorescent molecule, characterized in that The near-infrared second-region fluorescent molecule is shown in Formula I: Formula I; Wherein, R is hydroxy-C 1-6 alkyl.

2. A method for preparing a near-infrared second region fluorescent molecule, characterized in that: The preparation method comprises the following steps: S1: Sodium hydride, phenothiazine, and 3-bromopropanol are added to N,N-dimethylformamide for a substitution reaction, and the intermediate 1 represented by formula II is obtained by purification by column chromatography; Formula II; S2: Intermediate 1, N-bromosuccinimide and tetrahydrofuran are mixed and subjected to substitution reaction, and then purified by column chromatography to obtain intermediate 2 as shown in formula III; Formula III; S3: Intermediate 2, bis(pyrrolidino)diboron, potassium acetate, 1,4-dioxane and palladium catalyst are mixed and subjected to Suzuki coupling reaction, and purified by column chromatography to obtain intermediate 3 as shown in formula IV; Formula IV; S4: Intermediate 3, (2-bromoethylene-1,1,2-triyl)triphenyl, 4,8-bis(5-bromo-3-(2-ethylhexyl)-2-thienyl)-2λ4δ2-benzo[1,2-c:4,5-c']bis[1,2,5]thiadiazole, palladium catalyst and potassium carbonate are mixed and subjected to Suzuki coupling reaction, and the mixture is purified by column chromatography to obtain the near-infrared second-region fluorescent molecule BPT as shown in Formula V; Formula V.

3. The preparation method according to claim 2, characterized in that The molar ratio of sodium hydride, phenothiazine and 3-bromopropanol in step S1 is (1-4):1:1; In step S2, the molar ratio of intermediate 1 to N-bromosuccinimide is 1:(1-4), and the substitution reaction time is 16-32 h; In step S3, the molar ratio of the intermediate 2, bis(pyrrolidino)diboron, potassium acetate, and palladium catalyst is 1:(2-4):(4-8):(0.5-1), and the molar volume ratio of the intermediate 2 to 1,4-dioxane is 1:(2-4) mmol / mL; The reaction temperature of the Suzuki coupling reaction in step S3 is 70-90° C., and the reaction time is 30-42 h; In step S4, the molar ratio of intermediate 3, (2-bromoethylene-1,1,2-triyl)triphenyl, 4,8-bis(5-bromo-3-(2-ethylhexyl)-2-thienyl)-2λ4δ2-benzo[1,2-c:4,5-c']bis[1,2,5]thiadiazole, palladium catalyst and potassium carbonate is (20-30):1:(6-8):1:(30-40); The reaction temperature of the Suzuki coupling reaction in step S4 is 60-80° C., and the reaction time is 16-32 h.

4. A composition, characterized in that The composition comprises the near-infrared second region fluorescent molecule according to claim 1, a nitric oxide prodrug and an amphiphilic polymer carrier; The mass ratio of the near-infrared second-region fluorescent molecule, the nitric oxide prodrug and the amphiphilic polymer carrier is (0.5-2): (0.02-0.1):

10.

5. The composition according to claim 4, characterized in that The nitric oxide prodrug is an azodiol alkene salt compound.

6. The composition according to claim 5, characterized in that The azodicarbonium salt compound is O2-(2,4-dinitrophenyl)-1-[(4-ethoxycarbonyl)piperazinyl]diazenium-1,2-diol ester.

7. The composition according to claim 4, characterized in that The amphiphilic polymer carrier is selected from at least one of distearoylphosphatidylethanolamine-polyethylene glycol, polylactic acid-glycolic acid copolymer and polyoxyethylene polyoxypropylene ether triblock copolymer.

8. A method for preparing the composition according to any one of claims 4 to 7, characterized in that: The preparation method comprises the following steps: The near-infrared second-zone fluorescent molecule, nitric oxide prodrug, and amphiphilic polymer carrier are dissolved in an organic solvent to obtain a mixed solution; the mixed solution is added to water under ultrasonic treatment for self-assembly, and the organic solvent is removed to obtain a composition.

9. The preparation method according to claim 8, characterized in that The organic solvent is tetrahydrofuran; The mixing volume ratio of the mixed liquid to water is 1:(7-12).

10. Use of the near-infrared second region fluorescent molecule according to claim 1 or the composition according to any one of claims 4 to 7 in the preparation of a fluorescent probe; The fluorescent probe includes a fluorescent probe for at least one of (1)-(2): (1) Tumor imaging; (2) Cardiovascular imaging.

11. Use of the composition according to any one of claims 4 to 7 in the preparation of a product for diagnosing and / or treating solid tumors; The product for diagnosing and / or treating solid tumors includes a photodynamic nano-drug delivery system.

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