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

Abstract

The invention discloses a near-infrared two-region fluorescent molecule as well as a preparation method and application thereof. The near-infrared two-region fluorescent molecule is prepared by conjugating a phenothiazine group with a non-planar tricyclic structure and a strong electron acceptor benzobithiadiazole core, wherein the phenothiazine group has strong electron supply capability and active oxygen generation capability; a tetraphenyl ethylene module is further introduced to endow AIE characteristics; the fluorescent molecule shows NIR-II fluorescence emission at 1083 nm, the fluorescence quantum yield is 1.53%, the photothermal conversion efficiency is as high as 63%, and the fluorescent molecule has I-type ROS generation ability, and can be used as a near-infrared two-region fluorescence imaging contrast agent to realize multi-mode imaging of living blood vessels and tumor parts and imaging-guided photothermal-photodynamic synergistic treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a near-infrared second-zone 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 usually 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) effect caused by strong π-π interactions in large conjugated systems. These defects significantly limit their application in tumor diagnosis and treatment. In contrast, organic small molecules with aggregation-induced emission (AIE) properties show significant advantages in tumor multimodal 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-rotation structure of AIE molecules achieves a controllable balance between radiative energy and non-radiative energy relaxation paths in the aggregated state, making a multifunctional diagnostic and therapeutic platform based on a single AIE molecule possible. In addition, AIE molecules are of great research significance due to their large Stokes shift, superior photostability, and ability to passively target tumors. At present, a variety of NIR-II AIE molecules have been successfully used for multimodal imaging and therapy. However, the design and synthesis of AIE molecules with high fluorescence quantum yield, high photothermal conversion efficiency and strong reactive oxygen generation ability still face key challenges. Solving these problems 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-zone fluorescent molecule, which has aggregation-induced emission (AIE) characteristics and can achieve efficient multimodal imaging.

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

[0006] The present invention also provides a composition.

[0007] The invention also provides a method for preparing the composition.

[0008] The present invention also proposes the application of the above-mentioned near-infrared second-zone fluorescent molecules.

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

[0010] According to the first aspect of the present invention, a near-infrared second-region fluorescent molecule is proposed, and the chemical structure of the near-infrared second-region fluorescent molecule is as follows:

[0011]

[0012] Wherein, R is selected from alkyl, hydroxy-C 1-6 Any of an alkyl group, a substituted or unsubstituted phenyl group;

[0013] Substituents of the substituted or unsubstituted phenyl group include a hydroxy group.

[0014] In some embodiments of the present invention, the R is hydroxypropyl.

[0015] According to a second aspect of the present invention, a method for preparing a near-infrared second-region fluorescent molecule is provided, the preparation method comprising the following steps:

[0016] S1: adding sodium hydride, phenothiazine and 3-bromopropanol to N,N-dimethylformamide for substitution reaction, and purifying by column chromatography to obtain the intermediate 1 shown in formula II;

[0017]

[0018] S2: The intermediate 1, N-bromosuccinimide and tetrahydrofuran are mixed and subjected to substitution reaction, and purified by column chromatography to obtain the intermediate 2 shown in formula III;

[0019]

[0020] S3: mixing the intermediate 2, bis(pyrrolidinoyl)diboron, potassium acetate, 1,4-dioxane and palladium catalyst to perform Suzuki coupling reaction, and purifying by column chromatography to obtain the intermediate 3 shown in formula IV;

[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 purified by column chromatography to obtain a near-infrared second-zone 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(pyrrolidinoyl)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]palladium(II) dichloride.

[0031] According to a third aspect of the present invention, a composition is provided, comprising the near-infrared second region fluorescent molecule, 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-zone 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 furanyl 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 olefinium salt compound.

[0037] In some embodiments of the present invention, the azodiol olefin 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 into water under ultrasonic treatment for self-assembly, and the organic solvent is removed to obtain a composite.

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

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

[0043] In some embodiments of the present invention, the mixing 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 the 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 prepared by conjugating a non-planar tricyclic phenothiazine group with strong electron supply capability and reactive oxygen species (ROS) generation capability with a strong electron acceptor benzobithiadiazole (BBTD) core, and further introducing a tetraphenylethylene (TPE) module to impart AIE properties; the fluorescent molecule exhibits NIR-II fluorescence emission at 1083 nm, a fluorescence quantum yield of 1.53%, a photothermal conversion efficiency of up to 63%, and type I ROS generation capability; 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 above-mentioned near-infrared second zone fluorescent molecule, wherein the composition co-encapsulates the above-mentioned fluorescent molecule and the nitric oxide precursor drug JS-K; JS-K is a tumor-inhibiting drug that can efficiently consume reduced glutathione (GSH) in tumor cells and release NO. The NO released by JSK reacts with superoxide free radicals (O 2-) efficiently binds in the tumor microenvironment to form peroxynitrite (ONOO - ), thereby further inducing cancer cell apoptosis; at 808nm laser (0.7W / cm 2 ) irradiation, the composition showed significant cytotoxic effects on tumor cells at low concentrations. The composition achieved high-quality multimodal imaging of blood vessels and tumor areas in vivo, while showing superior photodynamic therapy effects, achieving complete tumor ablation with only one treatment.

[0055] Additional aspects and advantages of the present invention will be set forth in part in, and in part will be apparent from, the description which follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0057] Figure 1 This is the hydrogen NMR spectrum of compound 1c prepared in Example 1 of the present invention;

[0058] Figure 2 This is the carbon NMR spectrum of compound 1c prepared in Example 1 of the present invention;

[0059] Figure 3 This is the NMR hydrogen spectrum of compound 2b prepared in Example 1 of the present invention;

[0060] Figure 4 This is the carbon NMR spectrum of compound 2b prepared in Example 1 of the present invention;

[0061] Figure 5 This is the NMR hydrogen spectrum of compound 3c prepared in Example 1 of the present invention;

[0062] Figure 6 The carbon NMR spectrum of compound 3c prepared in Example 1 of the present invention;

[0063] Figure 7 This is the result of fluorescence imaging of blood vessels in mice of BPT NP in the test example of the present invention;

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

[0065] Fig. 9 This is a graph showing the results of reactive oxygen species detection in 4T1 cells after treatment with JS-K-BPT NPs in the test example of the present invention;

[0066] Fig.10 This is a microscopic result of the detection of reactive oxygen species in 4T1 cells after treatment with JS-K-BPT NPs in the test example of the present invention, wherein the scale bar is 30 μm;

[0067] Fig.11 The cell survival rate results of 4T1 cells treated under different conditions in the test examples of the present invention are shown in Figure 1, wherein Figures AD correspond to the cell survival rates of groups (1) to (4), respectively;

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

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

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

[0071] Fig.15 This is a diagram showing the quantum yield measurement results of BPT NPs in the experimental examples of the present invention;

[0072] Fig.16 This is a linear fitting result diagram of the cooling period time of BPT NPs and the negative natural logarithm of the driving force temperature in the test example of the present invention. DETAILED DESCRIPTION

[0073] Embodiments of the present invention are described in detail below, examples of which 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 only used to explain the present invention, and cannot be understood as limiting the present invention.

[0074] In the description of the present invention, the description with reference to the terms "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 schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0075] Example 1 Synthesis of near-infrared second region fluorescent molecules

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

[0077]

[0078] Step 1:

[0079]

[0080] Under ice bath conditions, NaH (600 mg, 25 mmol) was suspended in DMF (15 mL), and then compound 1a phenothiazine (2.0 g, 10.0 mmol) and compound 1b 3-bromopropanol (10.0 mmol) were added to obtain a suspension. The suspension was stirred at room temperature overnight; after the reaction was completed and cooled to room temperature, a saturated sodium chloride aqueous solution was added; the organic layer was collected and extracted with dichloromethane, and dried over anhydrous Na2SO4. After evaporating the organic solvent, the crude product was purified by column chromatography to obtain the desired purple oily product compound 1c (0.85 g, yield 35%).

[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 (101MHz, DMSO-d6)δ150.00,132.79,132.29,128.70,127.62,120.98,63.29,48.61,34.78. 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 THF were added to a flask and stirred at room temperature for 24 hours. After the reaction was completed, silica gel was directly added, and THF was removed by rotary evaporation. The crude product was purified by column chromatography using petroleum ether as an eluent to obtain compound 2a.

[0085] The crude product 2a after preliminary purification was mixed with bis(pyrrolidinoyl)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), and then sealed with a sealing film. The reaction system was vented by three freeze-vacuum-thaw cycles to remove air. The reaction system was heated to 80 ° C and reacted for 36 hours. After the reaction was completed, it was cooled to room temperature and washed with water; the organic layer was collected and dried over anhydrous Na2SO4 to obtain a crude product. The crude product was further purified by column chromatography using a petroleum ether / dichloromethane mixed solvent as an eluent to finally obtain a light yellow solid product compound 2b with a yield of 30% (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 (101MHz, Chloroform-d) δ147.33, 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 shown in Figure 3 and Figure 4 shown.

[0087] Step 3:

[0088]

[0089] 4,8-bis(5-bromo-3-(2-ethylhexyl)-2-thienyl)-2λ4δ2-benzo[1,2-c:4,5-c']bis[1,2,5]thiadiazole (Compound 3b, 500 mg, 0.73 mmol), 3-(3,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborol-2-yl)-10H-phenothiazin-10-yl)propan-1-ol (Compound 2b, 837 mg, 1.8 3mmol), (2-bromoethylene-1,1,2-triyl)triphenyl (compound 3a, 84mg, 0.073mmol), tetrakis(triphenylphosphine)palladium (Pd(PPh3)4, 84mg, 0.073mmol) and potassium carbonate (K2CO3, 353mg, 2.56mmol) were added to a tetrahydrofuran / water mixed solvent (mixed volume ratio 5:1, 60mL), and heated at 70°C for 24 hours under a nitrogen atmosphere. After the reaction, the reaction system 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 obtained concentrate was purified by silica gel column chromatography (using dichloromethane and n-hexane as eluents) to obtain the target product, dark green solid compound 3c (510mg, yield 9%), that is, the near-infrared second-zone fluorescent molecule BPT.

[0090] The NMR results of BPT are as follows: 1 H NMR (400MHz, DMSO-d6) δ7.72–7.33(m,8H),7.29–6.48(m,36H),4.60(s,2H),3.87(s,4H),3.51(d,J=4.9H z, 4H), 1.80 (d, J = 6.9Hz, 4H), 1.23–1.09 (m, 4H), 0.99–0.61 (m, 18H), 0.50 (d, J = 6.3Hz, 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 spectrum 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] In this example, a composition (JS-K-BPTNP) was prepared based on the near-infrared second-zone fluorescent molecule BPT prepared in Example 1. The specific preparation method is as follows:

[0093] Under 180W, 40kHz ultrasonic treatment, 1mL of THF solution containing 1mg BPT, 50μg JS-K (purchased from Yuanye Biotechnology, T23314) and 10mg DSPE-mPEG2000 (purchased from Shenzhen Meiluo Technology Co., Ltd., 020805) was poured into 9mL of deionized water and maintained for 10 minutes of ultrasound. 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 of the aqueous solution was 1mg / mL.

[0094] Example 3

[0095] This example prepares a nanoparticle (BPTNP) based on the near-infrared second-zone 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 test 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 test methods and results are as follows:

[0098] 1. Fluorescence imaging of BPT NPs in mice

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

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

[0101] 2. NIR-II-excited 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 government and international guidelines for the care and use of laboratory animals of the Ministry of Science and Technology of the People's Republic of China and approved by the Animal Care and Utilization Committee of Southern University of Science and Technology (resolution number: SUST-JY202406114). All mice were purchased from 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 was 1.6 mg / mL) via the tail vein. Then, in vivo NIR-II fluorescence images were captured on a NIR-II in vivo imaging system with a 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.) at different time points (4, 12, 24, and 48 hours) after injection. The results are shown in Figure 2. Figure 8 As shown in A. In addition, after intravenous injection of BPT NPs, photoacoustic imaging was also performed at specified time intervals on a photoacoustic imaging system. The photoacoustic imaging system was based on a US commercial platform (Vantage-128-HF, Verasonics, USA) to build a PA / US dual-mode imaging system, using an adjustable 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 7ns, the laser repetition rate was 20Hz, and the central frequency of the linear transducer was 7.5MHz; the results are shown in 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. The results are 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 reactive oxygen species generation in 4T1 cells

[0107] In this test example, 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 photos were taken under 488 nm excitation light, and the fluorescence of the corresponding probe at 525 nm was measured 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 content of BPT was 5 μM and the content of JS-K 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 are shown in Fig. 9 A and Fig.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 are shown in Fig. 9 B and Fig.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 with or without 808 nm laser irradiation (0.7 W / cm 2 ) was taken and the fluorescence intensity was recorded. The results are shown in Fig. 9 C and Fig.10 shown.

[0111] Depend on Fig. 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 808nm laser irradiation, BPT exerts its type I reactive oxygen species generation function to produce 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] The 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 3 The cells were plated on a 96-well plate at a density of 10 cells / mL 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 for 6 h in the dark, the cells were exposed to 808 nm laser irradiation (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 (0, 2, 5, 10, 20, and 50 μM for BPT and 0, 0.4, 0.8, 2, 4, and 10 μM for JS-K). After incubation for 6 h in the dark, 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;

[0119] After the incubation of the above-mentioned groups (I)-(IV), the culture medium was discarded and the cells were washed three times with PBS; then, the cells were incubated in fresh FBS-free RPMI1640 culture medium supplemented with 10% CCK-8 for 1 hour in the dark; 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 live cells after different treatments to untreated control cells, and 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 the figure. Fig.11 shown.

[0120] Depend on Fig.11 It can be seen that, in the case of no laser treatment, cells treated with BPT NPs can only reduce cell viability under high concentration (50 μM) treatment, while after laser treatment, only 10 μM BPT NPs are needed to achieve the effect of 50 μM without laser treatment. Under the conditions of laser and 50 μM BPT NPs, 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 under high concentration treatment; and after administering JS-K-BPT NPs 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 much better than that of JS-K or BPT NPs 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 10 cells / mL and incubated for 24 hours. Subsequently, 4T1 cells were exposed to different treatments:

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

[0124] (b) Incubated with PBS for 6 h and then irradiated with 808 nm laser 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 treatments (a)-(f), 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 488nm, PI is 561nm; emission filter: Calcein AM is 500-550nm, PI is 600-700nm. The results are shown in the figure. Fig.12 shown.

[0130] Depend on Fig.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 NP (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, and then irradiate with 808 nm laser 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 with 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. Fig.13 As shown, Fig.13 The first row of flow cytometry plots from left to right corresponds to the results of ①-③, and the second row of flow cytometry plots from left to right corresponds to the results of ④-⑥; wherein the Q4 quadrant represents normal cells, and the Q2 quadrant represents late apoptotic cells.

[0140] Depend on Fig.13 It can be seen that under the conditions of laser and JS-K-BPT NP (JS-K 4 μM, BPT 20 μM), 4T1 cells undergo a large amount of apoptosis.

[0141] 5. Anti-tumor effects of nanoparticles

[0142] To investigate the in vivo antitumor efficacy of BPT NPs and JS-K-BPT NPs, 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 mice in the BPT NPs group 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 mice in the JSK-BPT NPs group 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 mice in the control group were given 100 μL of nanoparticles BPT NPs (BPT content of 1.6 mg / mL) prepared in Example 3 through the tail vein of the mice in the BPT NPs+light group, and 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 the 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, and 12 hours after injection, the tumor site was irradiated 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 2 ) / 2 to estimate the tumor volume; the results are as follows Fig.14 shown.

[0145] Depend on Fig.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 that in 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 solution with absorbance values ​​of 0.02, 0.04, 0.06, 0.08, and 0.1 was measured under the same conditions; and 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 Fig.15 shown.

[0150] Depend on Fig.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 intervals of 20 seconds 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 Fig.16 shown.

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

[0154] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A near-infrared second region fluorescent molecule, characterized in that: The near-infrared second-zone fluorescent molecule is shown in Formula I: Wherein, R is selected from alkyl, hydroxy-C 1-6 Alkyl or substituted or unsubstituted phenyl; Substituents of the substituted or unsubstituted phenyl group include a hydroxy group.

2. A method for preparing a near-infrared second-region fluorescent molecule, characterized in that: The preparation method comprises the following steps: S1: adding sodium hydride, phenothiazine and 3-bromopropanol to N,N-dimethylformamide for substitution reaction, and purifying by column chromatography to obtain the intermediate 1 shown in formula II; S2: The intermediate 1, N-bromosuccinimide and tetrahydrofuran are mixed and subjected to substitution reaction, and purified by column chromatography to obtain the intermediate 2 shown in formula III; S3: mixing the intermediate 2, bis(pyrrolidinoyl)diboron, potassium acetate, 1,4-dioxane and palladium catalyst to perform Suzuki coupling reaction, and purifying by column chromatography to obtain the intermediate 3 shown in 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 purified by column chromatography to obtain a near-infrared second-zone fluorescent molecule BPT as shown in 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; Preferably, in step S2, the molar ratio of the intermediate 1 to N-bromosuccinimide is 1:(1-4), and the substitution reaction time is 16-32h; Preferably, in step S3, the molar ratio of the intermediate 2, bis(pyrrolidinoyl)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; Preferably, the reaction temperature of the Suzuki coupling reaction in step S3 is 70-90° C., and the reaction time is 30-42 h; Preferably, in step S4, 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 is (20-30):1:(6-8):1:(30-40); Preferably, 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 as claimed in claim 1, a nitric oxide prodrug and an amphiphilic polymer carrier; The mass ratio of the near-infrared second-zone 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 includes at least one of an organic nitrate compound, a nitroprusside salt compound, an S-nitrosothiol compound, a furan nitrogen oxide compound, a hydroxamic acid compound, and an azodiol olefinium salt compound; Preferably, the nitric oxide prodrug is an azodiol olefin salt compound; More preferably, the azodicarbonium salt compound is O2-(2,4-dinitrophenyl)-1-[(4-ethoxycarbonyl)piperazinyl]diazenium-1,2-diol ester.

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

7. A method for preparing a composition as claimed in any one of claims 4 to 6, 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 into water under ultrasonic treatment for self-assembly, and the organic solvent is removed to obtain a composite.

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

9. Use of the near-infrared second region fluorescent molecule as claimed in claim 1 or the composition as claimed in any one of claims 4 to 6 in the preparation of a fluorescent probe; Preferably, the fluorescent probe comprises a fluorescent probe for at least one of (1)-(3): (1) Tumor imaging; (2) Cardiovascular imaging; (3)Neuroimaging.

10. Use of the composition according to any one of claims 4 to 6 in the preparation of a product for diagnosing and / or treating tumors; Preferably, the product for diagnosing and / or treating tumors comprises a photodynamic nano-drug delivery system.

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