Mitochondria-targeted fluorescent dyes and their applications in bioimaging and tumor therapy

Abstract

The application provides a near-infrared two-region fluorescent dye with mitochondrion targeting and aggregation-induced emission properties and application thereof in biological fluorescence imaging and tumor treatment. 41 H 37 F6N2PS, which has good light stability, outstanding mitochondrion targeting, clear ROS production mechanism and excellent photothermal performance. The substance has good mitochondrion targeting capacity and can destroy tumor mitochondria under laser irradiation of a certain intensity, thereby killing tumor cells. The aggregation-induced emission property makes it different from traditional aggregation quenching emission materials and has better biological imaging effect. The near-infrared property makes it have good tissue penetration capacity and biological fluorescence imaging effect. The substance has wide application prospects in near-infrared fluorescence imaging, preparation of mitochondrion targeting reagents and photodynamic therapy reagents. It can be applied to combined phototherapy of breast tumor PDT and PTT, and has simple synthesis steps and is conducive to commercial application and popularization.

Description

TECHNICAL FIELD

[0001] The application relates to a mitochondria-targeting near-infrared two-region AIE fluorescent dye with an aggregation-induced emission property and an application thereof, and belongs to the technical field of optical materials. BACKGROUND

[0002] Phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), is a rapidly developing cancer treatment that utilizes light and an absorber to generate cytotoxic reactive oxygen species (ROS) or local temperature enhancement in the target tissue. Unlike traditional treatment methods, phototherapy has the advantages of non-ionizing radiation, minimal invasiveness, low toxicity, and high spatiotemporal precision. These characteristics make phototherapy particularly suitable for the treatment of superficial and in situ tumors. During the treatment process of photodynamic therapy, ROS has two different mechanisms, including type I and type II pathways. Type I pathway does not depend on oxygen, and photosensitizer (PS) transfers electrons to substrates to form ROS, making it the preferred treatment for hypoxic tumors. Type II pathway depends on oxygen, and PS interacts with oxygen to produce highly toxic singlet oxygen.

[0003] So far, various types of photodynamic therapy studies have adopted various photosensitizers, including porphyrins, phthalocyanines, and phenothiazines. However, due to the aggregation-caused quenching (ACQ) effect, the performance and treatment ability of traditional photosensitizers are greatly limited. Aggregation-induced emission (AIE) materials, on the other hand, have overcome the quenching disadvantage of traditional materials. Due to their inherent photophysical characteristics, AIEgens have good compatibility, high luminescence efficiency, and strong active oxygen production ability in the aggregated state. Therefore, compared to traditional photosensitizers, AIE materials have a unique advantage in tumor phototherapy.

[0004] Mitochondria are essential organelles in eukaryotic cells that produce energy in the form of adenosine triphosphate and control programmed cell death through the balance of anti-apoptotic and pro-apoptotic proteins. Mitochondria are closely related to the redox state of the cell. Cancer cells rely heavily on mitochondria to maintain their rapid energy consumption and support their growth and survival. Many studies have been conducted on mitochondria-targeting drugs, and phototherapy targeting mitochondria may provide a new option for tumor treatment. Due to the role of mitochondria in cell activity and function, it is an ideal target for tumor treatment.

[0005] To date, a variety of organic small-molecule mitochondrial targeting agents with various structures have been developed, including derivatives of BODIPY, coumarin, cyanine, xanthene, indole, etc. However, most of these mitochondrial targeting dyes are usually in the visible or NIR-I region, which exhibit relatively short fluorescence emission, while mitochondrial targeting dyes emitting in the NIR-II region are significantly scarce. Due to their small Stokes shift and relatively short emission wavelength, their biomedical applications are limited to some extent. Traditional photosensitizers can be activated by short-wavelength light and have been successfully applied to superficial cancers, such as skin and esophageal tumors. However, it is still challenging for deeper tumors. Near-infrared region II (NIR-II) refers to the spectral region with a wavelength of 1000-1700 nm. The penetration depth of long-wavelength near-infrared (NIR) light is significantly higher than that of short-wavelength ultraviolet and visible light, so NIR light in the NIR-II is considered to be the preferred phototherapy method for eliminating deep tumors because it has the advantages of higher maximum permissible exposure, deeper penetration, and low autofluorescence. Recently, there have been reports that a variety of traditional organic fluorescent dyes and AIE dyes exhibiting NIR-II fluorescence have been used in biological imaging and biomedical applications. However, most of these dyes cannot effectively target mitochondria, which may be due to their non-ionic molecular structure and excessively high molecular weight.

[0006] Combination phototherapies, including PDT and PTT, have great potential to achieve local tumor destruction. However, their biomedical applications also face challenges. Most PSs are based on the O2-dependent type II PDT mechanism, which involves energy transfer between the excited-state PS and endogenous O2 in tumors, thereby generating cytotoxic O2. Unlike these O2-dependent type II PSs, type I PSs do not depend on O2 and have better antitumor effects for hypoxic tumor microenvironments.

[0007] Therefore, it is of great significance to develop AIE luminescent materials in the near-infrared region II with mitochondrial targeting properties. SUMMARY

[0008] The present application overcomes the problems in the background art and provides an AIE luminescent material in the near-infrared region II with mitochondrial targeting properties and a preparation method thereof.

[0009] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0010] A mitochondrial-targeting near-infrared region II fluorescent dye with aggregation-induced emission properties, whose molecular formula is: 41 H 37 F6N2PS, and the structural formula is

[0011] .

[0012] A preparation method of a mitochondria-targeting near-infrared two-region fluorescent dye with an aggregation-induced emission property, comprising the following steps:

[0013] 5-(4-(di-p-tolylamino)phenyl)thiophene-2-carboxaldehyde, 1,1,2,3-tetramethyl-1H-benzo[e]indolium hexafluorophosphate, and 3-methylpiperidine are mixed, an appropriate amount of anhydrous ethanol is added, stirring is performed under a nitrogen atmosphere at room temperature for 48 hours, the solvent is evaporated under reduced pressure, elution is performed using dichloromethane / methanol as an eluent, and the target product is obtained through silica gel column chromatography purification.

[0014] The molar ratio of 5-(4-(di-p-tolylamino)phenyl)thiophene-2-carboxaldehyde and 1,1,2,3-tetramethyl-1H-benzo[e]indolium hexafluorophosphate is 1:1.

[0015] A mitochondria-targeting near-infrared fluorescent dye BIH-TB has a molecular formula of C 27 H 24 F6NPS, and a structural formula of:

[0016] .

[0017] A preparation method of a fluorescent dye BIH-TB with an aggregation-induced emission property, 5-phenylthiophene-2-carboxaldehyde, 1,1,2,3-tetramethyl-1H-benzo[e]indolium hexafluorophosphate, 3-methylpiperidine, and anhydrous ethanol are mixed, stirring is performed under a nitrogen atmosphere at room temperature for 48 hours, the solvent is evaporated under reduced pressure, and then elution is performed through silica gel column chromatography purification using dichloromethane / methanol as an eluent to obtain a solid powder.

[0018] The molar ratio of 5-phenylthiophene-2-carboxaldehyde and 1,1,2,3-tetramethyl-1H-benzo[e]indolium hexafluorophosphate is 1:1.

[0019] The mitochondria-targeting near-infrared fluorescent dye is applied to near-infrared fluorescence imaging.

[0020] The mitochondria-targeting near-infrared two-region fluorescent dye with an aggregation-induced emission property is applied to preparation of a mitochondria-targeting reagent.

[0021] The mitochondria-targeting near-infrared two-region fluorescent dye with an aggregation-induced emission property is applied to preparation of a phototherapy tumor preparation.

[0022] The fluorescent dye BIH-TB is applied to preparation of a mitochondria-targeting reagent.

[0023] The fluorescent dye BIH-TB is applied to preparation of a phototherapy tumor preparation.

[0024] 1. The near-infrared second region (NIR-II) luminescent dye BIH-TA of the present application has both mitochondrial targeting properties and aggregation-induced emission (AIE) properties, and has the following advantages compared with traditional photosensitizers:

[0025] (1) Good light stability, outstanding mitochondrial targeting property, clear ROS production mechanism, excellent photothermal performance, and large stoke shift.

[0026] (2) The near-infrared second region aggregation-induced emission dye of the present application has good mitochondrial targeting ability and near-infrared bioimaging ability. Targeting mitochondria enables it to effectively destroy tumor mitochondria and thus kill tumor cells. The aggregation-induced emission property distinguishes it from traditional aggregation quenching emission materials, and it has better bioimaging effect. The near-infrared property enables it to have good tissue penetration ability and bioimaging effect. It has wide application prospects in near-infrared fluorescence imaging, mitochondrial targeting reagent preparation, and photodynamic therapy reagents.

[0027] (3) BIH-TA of the present application shows strong NIR-II fluorescence, has typical AIE characteristics and significant mitochondrial targeting properties, and can be effectively applied to breast tumor PDT and PTT combined therapy to achieve good antitumor effect. Importantly, synergistically inducing apoptosis and pyroptosis elucidates the molecular mechanism of combined phototherapy. These positive results open up potential applications of multifunctional NIR-II AIE materials in breast tumor combined phototherapy.

[0028] (4) The synthesis steps of the present application are simple, which is conducive to commercial application and popularization.

[0029] At room temperature, ethanol is used as an organic solvent, and 3-methylpiperidine is used as an alkaline catalyst to prepare by Knoevenagel condensation reaction. The chemical structure of the product is characterized by using standard spectroscopic analysis technology and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS), which confirms that the chemical structure of the product is correct and the purity is high.

[0030] 2. The present application also provides another near-infrared fluorescent dye BIH-TB with mitochondrial targeting properties, which emits bright fluorescence in the red and NIR regions, with an emission peak at 678 nm and an absorption wavelength peak at 486 nm. The material is simple to prepare and easy to implement. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is the normalized fluorescence spectrum of BIH-TA and BIH-TB;

[0032] Figure 2 is the relative fluorescence intensity (I / I0) of BIH-TA and BIH-TB; I / I 0) Relationship of BIH-TA with tetrahydrofuran / water mixture components;

[0033] Figure 3 Temperature change of BIH-TA under different power laser irradiation;

[0034] Figure 4 Active oxygen generation of BIH-TA after laser irradiation;

[0035] Figure 5 Hydroxyl radical generation of product after laser irradiation.

[0036] Figure 6 Co-localization imaging of BIH-TA stained U2-OS cells and commercial mitochondrial probe;

[0037] Figure 7 Comparison of light stability of BIH-TA and commercial green mitochondrial probe in U2-OS cells obtained by laser confocal microscope continuous scanning;

[0038] Figure 8 Comparison of relative signal intensity of BIH-TA and commercial green mitochondrial probe in U2-OS cells under laser continuous scanning;

[0039] Figure 9 Comparison of U2-OS cell toxicity under 660 nm laser irradiation and darkness after BIH-TA incubation;

[0040] Figure 10 Confocal fluorescence images of Calcein-AM and PI of U2-OS cells after incubation with BIH-TA in different experimental groups;

[0041] Figure 11 Relative tumor volume-time curve of different experimental groups of mouse tumors;

[0042] Figure 12 BIH-TA effect on mouse tumor weight change chart;

[0043] Figure 13 NIR-II fluorescence images of different monitoring time points after injection of BIH-TA into mouse orthotopic tumors;

[0044] Figure 14 Average fluorescence intensity-time curve in Figure 13

[0045] Signal-to-background ratio-time curve in Figure 15 Figure 13

[0046] Figure 16 ​​The in vitro NIR-II fluorescence images of different organs of mice injected with BIH-TA 48 h later. DETAILED DESCRIPTION

[0047] The application will be further described in detail below in conjunction with the examples. Unless otherwise specified, the instruments and equipment involved in the examples are all conventional instruments and equipment; the reagents involved are all commercially available conventional reagents; and the test methods involved are all conventional methods.

[0048] Example 1 Preparation method of mitochondria-targeting near-infrared light-emitting agent with aggregation-induced emission

[0049] The mitochondria-targeting near-infrared light-emitting material (BIH-TA for short) has a molecular formula of C 41 H 37 F6N2PS, a molecular weight of 734.79, and a structural formula of:

[0050]

[0051] The synthesis route is as follows:

[0052]

[0053] Among them,

[0054]

[0055] The preparation of BIH-TA is by Knoevenagel condensation reaction with ethanol as the organic solvent and 3-methylpiperidine as the basic catalyst to obtain the target product.

[0056] Specific method: 5-(4-(di-p-tolylamino)phenyl)thiophene-2-carboxaldehyde 766 mg (2 mmol), 1,1,2,3-tetramethyl-1H-benzo[e]indole hexafluorophosphate 738 mg (2 mmol), 3-methylpiperidine 0.5 mL, and anhydrous ethanol 20 mL were mixed, stirred at room temperature under a nitrogen atmosphere for 48 hours, the solvent was evaporated under reduced pressure, and then purified by silica gel column chromatography with dichloromethane / methanol solution as the eluent to obtain a solid powder 811 mg with a yield of 55.2%.

[0057] The chemical structure of the obtained product was characterized, and the following data were obtained:

[0058] 1 H NMR (500 MHz, dichloromethane- d 2) δ 8.35 (d, J = 15.4 Hz, 1H), 8.23 (d, J =8.5 Hz, 1H), 8.14 (d,J = 8.8 Hz, 1H), 8.08 (d, J = 8.2 Hz, 1H), 7.82 (d, J = 4.2Hz, 1H), 7.76 (t, J = 7.2 Hz, 1H), 7.69–7.65 (m, 2H), 7.57 (d, J = 8.7 Hz, 2H), 7.45 (d, J = 4.2 Hz, 1H), 7.16 (d, J = 7.9 Hz, 4H), 7.06 (d, J = 8.3 Hz, 4H), 6.97(d, J = 8.7 Hz, 2H), 6.90 (d, J = 15.4 Hz, 1H), 4.09 (s, 3H), 2.35 (s, 6H), 2.05 (s, 6H).

[0059] 13 C NMR (126 MHz, dichloromethane-) d 2) δ 181.24, 158.32, 151.06, 145.44,144.24, 141.51, 139.33, 138.18, 137.65, 135.12, 133.96, 132.05, 130.68,130.63, 128.99, 127.97, 127.85, 127.63, 126.33, 125.22, 124.37, 123.13,120.33, 111.89, 107.19, 34.30, 26.95, 21.04. The peak of the methyl carbon atom connected to the positively charged nitrogen atom overlapped with the solvent signal (53.41–54.21 ppm), therefore the solvent used in subsequent tests was changed to DMSO. d 6(dimethyl sulfoxide-) d 6). 13 CNMR (151 MHz, DMSO- d6) δ 180.95, 154.01, 149.29, 144.32, 143.68, 139.80, 139.50, 138.10, 137.38, 133.80, 132.93, 130.80, 130.34, 130.01, 128.33, 127.31, 126.86, 126.73, 125.51, 125.01, 124.38, 123.04, 120.03, 113.02, 109.00, 53.20, 34.23, 25.39, 20.45. 19 F NMR (470 MHz, DMSO- d 6) δ -69.70 (d, J =709.7 Hz), 31 P NMR (202 MHz, DMSO- d 6) δ -144.14 (sept, J = 711.0). FTMS (APCIsource) m / z: [M-PF6] + calcd for C 41 H 37 N2S + , 589.2672; found, 589.2672。

[0060] Analysis shows that the synthesis method of the application obtains the correct target product BIH-TA.

[0061] Example 2 Preparation method of mitochondria-targeting fluorescent dye BIH-TB

[0062] The mitochondria-targeting fluorescent dye BIH-TB (referred to as BIH-TB) has a molecular formula of C 27 H 24 F6NPS and a molecular weight of 539.52, and its structural formula is as follows:

[0063]

[0064] Preparation of the BIH-TB compound:

[0065] A mixture of 5-phenylthiophene-2-carboxaldehyde 3b (376 mg, 2 mmol), 1,1,2,3-tetramethyl-1H-benzo[e]indolium hexafluorophosphate 4 (738 mg, 2 mmol), 3-methylpiperidine (0.5 mL) and absolute ethanol (20 mL) was stirred at room temperature under nitrogen atmosphere for 48 hours. After evaporation of the solvent under reduced pressure, the product was purified by column chromatography on silica gel eluting with dichloromethane / methanol as eluent to obtain a solid powder (558 mg) with a yield of 51.8%.

[0066] The chemical structure of the obtained product was characterized, obtaining the following data:

[0067] 1 H NMR (500 MHz, DMSO- d 6) δ 8.75 (d, J = 16.0 Hz, 1H), 8.44 (d, J = 8.4 Hz,1H), 8.29 (d, J = 8.9 Hz, 1H), 8.23–8.20 (m, 2H), 8.10 (d, J = 9.0 Hz, 1H), 7.88–7.85 (m, 3H), 7.82 (ddd, J = 8.3, 6.8, 1.4 Hz, 1H), 7.73 (t, J = 7.5 Hz, 1H),7.55 (t, J = 7.5 Hz, 2H), 7.48 (t, J = 7.3 Hz, 1H), 7.35 (d, J = 16.0 Hz, 1H), 4.23(s, 3H), 2.02 (s, 6H). 13 C NMR (151 MHz, DMSO- d6) δ 181.40 (s, 1C), 152.63 (s,1C), 144.19 (s, 1C), 139.47 (s, 2C), 138.67 (s, 1C), 137.77 (s, 1C), 133.07(s, 1C), 132.51(s, 1C), 130.87 (s, 1C), 130.03 (s, 1C), 129.71 (s, 1C), 129.54 (s, 2C), 128.39 (s, 1C), 127.05 (s, 1C), 126.69 (s, 1C), 126.46 (s,1C), 126.06 (s, 2C), 123.13 (s, 1C), 113.14 (s, 1C), 110.32 (s, 1C), 53.45 (s, 1C), 34.53 (s, 1C), 25.23 (s, 2C). 19 F NMR (470 MHz, DMSO- d 6) δ -73.23 (d, J = 705.0 Hz). 31 P NMR (202 MHz, DMSO- d 6) δ -144.14 (sept, J = 711.0).

[0068] FTMS (APCI source) m / z [M-PF6] + calcd for C 27 H 24 NS + , 394.1624; found, 394.1627.

[0069] Analysis shows that the synthesis method of the present invention yielded the target product BIH-TB.

[0070] Example 3: Uses and performance of the mitochondrial-targeting fluorescent dye of the present invention

[0071] (1) Normalized absorption and fluorescence spectroscopy detection

[0072] Normalized fluorescence spectra of the products in Examples 1 and 2 are shown below. Figure 1 .

[0073] from Figure 1It can be seen that BIH-TA has a maximum emission peak at 856 nm in DMSO, and the emission tail extends to the NIR-II region beyond 1000 nm.

[0074] BIH-TA exhibits a large Stokes shift and emits NIR-II fluorescence, which is ideal for high-contrast NIR-II fluorescence imaging applications. NIR-II fluorescence has the advantages of small light scattering, low autofluorescence, and strong deep tissue penetration depth.

[0075] From Figure 1 It can also be seen that BIH-TB emits bright fluorescence in the red and NIR regions, with an emission peak at 678 nm and an absorption wavelength peak at 486 nm.

[0076] (2) Fluorescence spectrum of the product

[0077] Figure 2 The relative fluorescence intensity (I / I0) is plotted against the THF / water mixture composition of BTA. I / I 0) and BTA THF / water mixture composition. I 0=emission intensity in pure THF.

[0078] The fluorescence spectrum of the product BIH-TA in different water contents (vol%) of tetrahydrofuran / water mixture is shown in f w , where the solution concentration of BIH-TA is 0.1 mg / mL, the excitation wavelength is 585 nm, and the water content in the tetrahydrofuran / water mixture is represented by Figure 2 (vol%). f w

[0079] BIH-TA emits weak light in pure tetrahydrofuran solution. At the same concentration, as the water content in the tetrahydrofuran / water mixture increases (10-60%), the solution fluorescence intensity decreases. With further increase of water content (70-90%), the fluorescence intensity of the solution increases significantly. When the water content reaches 90%, the fluorescence of the solution reaches a maximum. This is due to the aggregation of BIH-TA molecules caused by the addition of solvent water, resulting in fluorescence enhancement, indicating that BIH-TA has excellent aggregation-induced emission properties.

[0080] (3) Temperature change of the product under different power laser irradiation

[0081] Figure 3 The temperature change of BIH-TA under different power laser irradiation at a concentration of 200 μM and a wavelength of 660 nm.

[0082] ​It can be seen that the temperature of BIH-TA with a concentration of 200 μM increases with the increase of power when irradiated by laser with different powers; when the power is constant, the temperature increases with the increase of laser irradiation time, and the temperature increases more obviously at a higher power, indicating that BIH-TA has excellent photothermal performance.

[0083] (4) Generation of reactive oxygen species after laser irradiation of the product

[0084] The generation of reactive oxygen species after 660 nm laser irradiation was observed using DCFH-DA (2',7'-dichlorofluorescein diacetate) as an indicator, as shown in Figure 4 .

[0085] DCFH-DA emits fluorescence when it reacts with reactive oxygen species. When a solution without BIH-TA was irradiated by 660 nm laser (1 W / cm²), the fluorescence intensity changed little, while when a DCFH-DA solution containing BIH-TA was irradiated by 660 nm laser (1 W / cm²), the fluorescence intensity of the solution increased significantly, indicating that BIH-TA generates a large amount of reactive oxygen species under 660 nm laser irradiation. The increase of reactive oxygen species in tumor cells helps to kill tumor cells.

[0086] The generation of hydroxyl radicals after 660 nm laser irradiation was observed using HPF (hydroxyphenyl fluorescein) as an indicator, as shown in Figure 5 .

[0087] HPF emits fluorescence when it reacts with hydroxyl radicals. When a solution without BIH-TA was irradiated by 660 nm laser (1 W / cm²), the fluorescence intensity changed little, while when a HPF solution containing BIH-TA was irradiated by 660 nm laser (1 W / cm²), the fluorescence intensity of the solution increased significantly, indicating that BIH-TA generates a large amount of hydroxyl radicals under 660 nm laser irradiation. This indicates that BIH-TA can generate reactive oxygen species through Type I pathway.

[0088] (5) Co-localization imaging of BIH-TA stained U2-OS cells and commercial mitochondrial probes, as shown in Figure 6 , where A: BIH-TA; B: commercial green mitochondrial probe; C: overlap; D: co-localization coefficient.

[0089] U2-OS cells were stained with commercial green mitochondrial probes and BIH-TA at 37°C for 30 minutes for co-localization imaging, and the specific method was as follows:

[0090] The BIH-TA with a concentration of 1 μM and the commercial green mitochondrial probe with a concentration of 1 μM were respectively incubated with U2-OS cells for 30 minutes, and after imaging under a confocal microscope, co-localization was performed. It was found that the co-localization coefficient (Pierce coefficient) was as high as 0.91, indicating that the BIH-TA could target the mitochondria of cells.

[0091] (6) Comparison of the light stability data of BIH-TA and the commercial green mitochondrial probe in U2-OS cells.

[0092] Figure 7 Comparison of the light stability images of BIH-TA and the commercial green mitochondrial probe in U2-OS cells scanned by a laser confocal microscope.

[0093] From Figure 7 It can be seen that, compared with the commercial mitochondrial material, the BIH-TA has more excellent light stability.

[0094] Comparison of the relative signal intensity of BIH-TA and the commercial green mitochondrial probe in U2-OS cells under laser continuous scanning, see Figure 8 .

[0095] The BIH-TA (1 μM) and the commercial green mitochondrial probe (1 μM) were respectively incubated with U2-OS cells for 30 minutes, and then they were respectively scanned for 90 times by using the same power laser. It was found that the relative signal intensity of the BIH-TA decreased less, while that of the commercial green mitochondrial probe decreased obviously, indicating that the BIH-TA has good light stability.

[0096] (7) Effect of BIH-TA with different concentrations on the cytotoxicity of U2-OS cells under laser irradiation

[0097] Comparison of the cytotoxicity of U2-OS cells after BIH-TA incubation under 660 nm laser irradiation and in the dark, see Figure 9 .

[0098] U2-OS cells were plated in a 96-well plate at 10,000 cells per well. After the cells adhered, BIH-TA solutions with concentrations of 0.1 μM, 0.25 μM, 0.5 μM, 0.75 μM and 1 μM were added to the 96-well plate, respectively, and incubated for 12 hours. Then, the cells were treated in the dark and irradiated by 660 nm laser (0.3 W / cm²) for 30 minutes, respectively. After waiting for 12 hours, the cytotoxicity was tested by MTT method.

[0099] Under dark conditions, the BIH-TA had little toxicity to cells within the tested concentration range. However, after irradiation by 660 nm laser, the cytotoxicity of the cells increased with the increase of the concentration of BIH-TA, indicating that the BIH-TA has excellent phototoxicity.

[0100] (8) Confocal fluorescence images of Calcein-AM and PI of U2-OS cells after incubation with BIH-TA

[0101] U2-OS cells were plated in confocal dishes and when they reached 80% confluence, they were divided into four groups, namely PBS group, PBS + laser group (660 nm, 0.3 W, 30 min), BIH-TA (1 μM) group and BIH-TA + laser group (660 nm, 0.3 W, 30 min).

[0102] Subsequently, cell staining was performed using Calcein-AM and PI, and confocal microscopy was used for imaging. If the cell is alive, it emits green fluorescence; if the cell is dead, it emits red fluorescence. The results show that only the BIH-TA + laser group is red, indicating that a large number of tumor cells in this group were killed, and the other three groups of cells are in good activity, indicating that BIH-TA has excellent phototoxicity.

[0103] Figure 10 Confocal fluorescence images of Calcein-AM and PI of U2-OS cells after incubation with BIH-TA, where A is the PBS group, B is the PBS + laser group, C is the BIH-TA group, and D is the BIH-TA + laser group. The scale bar is 100 μm.

[0104] (9) Animal experiment of BIH-TA on tumor growth

[0105] Balb / c mice breast cancer subcutaneous tumor models were established using 4T1 tumor cells. The experimental design included randomizing 4T1-bearing mice into four groups, (I) PBS group, (II) PBS + laser group, (III) BIH-TA group and (IV) BIH-TA + laser group, with seven mice in each group.

[0106] (I) The PBS group only received PBS injection, (II) the group received PBS injection at the same time as laser treatment. (III) The group only received BIH-TA treatment, (IV) The group received BIH-TA treatment at the same time as laser treatment once, with a laser power of 0.5 Wcm, a wavelength of 660 nm, and a laser irradiation time of 10 minutes. The mice's body weight, tumor weight and tumor volume were evaluated every two days for 12 days.

[0107] During the entire treatment period, the body weight of all groups of mice increased slightly and remained basically consistent, the tumor size of mice in (I), (II) and (III) groups increased significantly, and by the 12th day, the tumor volume increased by 13 times, 14 times and 12 times, respectively. In contrast, the tumor volume of the BIH-TA + laser group (IV) remained basically unchanged, and stabilized at 150 mm 3The weight of the right group was significantly reduced, indicating that the tumor growth of this group was significantly inhibited, see Figure 11 , Figure 12 .

[0108] (10) Tumor fluorescence imaging ability of BIH-TA

[0109] To study the tumor fluorescence imaging ability of BIH-TA, in situ 4T1 tumor-bearing mice were injected with BIH-TA solution, and then the whole-body NIR-II fluorescence images were collected over time ( Figure 13 ). Strong NIR-II fluorescence was observed immediately from BIH-TA, with a high signal-to-background ratio (SBR) of about 26. The emission intensity of the tumor region rapidly decreased to about 30% of the initial value 72 h after injection ( Figure 14 ). The quantitative value of SBR followed a similar trend, decreasing from 26 to 7 72 h after injection ( Figure 15 ).

[0110] The ex vivo NIR-II fluorescence images of the excised tumors and different organs of 4T1 xenograft mice 48 h after injection of BIH-TA ( Figure 16 ) further showed that BIH-TA mainly existed in the tumor site, and partially existed in the liver and spleen, which may be related to the continuous metabolism in the mouse body.

Claims

1. A mitochondrial-targeting near-infrared fluorescent dye BIH-TB, characterized in that, The molecular formula of the mitochondrial-targeted near-infrared fluorescent dye BIH-TB is: C 27 H 24 F6NPS, the structural formula is: 。 2. A method for preparing the mitochondrial-targeting near-infrared fluorescent dye BIH-TB as described in claim 1, characterized in that, 5-Phenythiophene-2-carboxaldehyde, 1,1,2,3-tetramethyl-1H-benzo[e]indole hexafluorophosphate, 3-methylpiperidine and anhydrous ethanol were mixed and stirred at room temperature for 48 hours under nitrogen atmosphere. The solvent was evaporated under reduced pressure and then purified by silica gel column chromatography using dichloromethane / methanol as eluent to obtain a solid powder. The molar ratio of 5-phenylthiophene-2-carboxaldehyde to 1,1,2,3-tetramethyl-1H-benzo[e]indole hexafluorophosphate is 1:

1.

3. The use of the fluorescent dye BIH-TB according to claim 1 in the preparation of mitochondrial targeting reagents.

4. The use of the fluorescent dye BIH-TB according to claim 1 in the preparation of phototherapy tumor agents.

Images