Mitochondria-targeted fluorescent dye and application thereof in biological imaging and tumor treatment

Abstract

The invention provides a near-infrared two-region fluorescent dye which is targeted to mitochondria and has aggregation-induced emission property and application of the near-infrared two-region fluorescent dye in biological fluorescence imaging and tumor treatment. The near-infrared two-region luminescent material BIH-TA with the mitochondrial targeting and aggregation-induced emission characteristics has a molecular formula of C41H37F6N2PS, and has the advantages of good light stability, prominent mitochondrial targeting, clear ROS production mechanism and excellent photo-thermal performance. The substance has good mitochondrial targeting ability, and can destroy tumor mitochondria under irradiation of laser with certain intensity so as to kill tumor cells. Due to the aggregation-induced emission property, the material is different from a traditional aggregation-quenching luminescent material, and a better biological imaging effect is achieved. Due to the near-infrared characteristic, the material has good tissue penetrating power and biological fluorescence imaging effect. The substance has a wide application prospect in near-infrared fluorescence imaging, preparation of mitochondrial targeting reagents and photodynamic therapy reagents. The compound can be applied to combined phototherapy of breast tumors PDT and PTT, the synthesis steps are simple, and commercial application and popularization are facilitated.

Description

Technical Field

[0001] The invention relates to a mitochondrial-targeted near-infrared zone II AIE fluorescent dye with aggregation-induced emission properties and application thereof, belonging to the technical field of optical materials. Background Art

[0002] Phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), is a rapidly developing cancer treatment modality that uses light and absorbers to generate cytotoxic reactive oxygen species (ROS) or local temperature enhancement in target tissues. Unlike traditional treatments, phototherapy has the advantages of non-ionizing radiation, minimal invasiveness, low toxicity, and high temporal and spatial precision. These characteristics make phototherapy particularly suitable for the treatment of superficial and in situ tumors. During the treatment of photodynamic therapy, there are two different mechanisms for ROS, including type I and type II pathways. The type I pathway is independent of oxygen, and the photosensitizer (PS) transfers electrons to the substrate to form ROS, making it the first choice for treating hypoxic tumors. The type II pathway is dependent on oxygen, and PS interacts with oxygen to produce highly toxic singlet oxygen.

[0003] So far, various types of photodynamic therapy studies have used a variety of photosensitizers, including porphyrins, phthalocyanines, and phenothiazines. However, due to the aggregation-induced quenching (ACQ) effect, the performance and therapeutic ability of traditional photosensitizers are greatly limited. Aggregation-induced emission (AIE) materials have well avoided the disadvantage of easy quenching of traditional materials. Due to their inherent photophysical characteristics, AIE luminophores (AIEgens) have the advantages of good compatibility, high luminescence efficiency, and strong ability to produce reactive oxygen in the aggregated state. Therefore, compared with traditional photosensitizers, AIE materials have unique advantages 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 cells. Cancer cells rely heavily on mitochondria to maintain their rapid energy consumption and support their growth and survival. Many studies on targeted drugs for mitochondria have been carried out, and phototherapy targeting mitochondria may provide a new option for tumor treatment. Due to the role of mitochondria in cell activities and functions, 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 light or NIR-I region, and they 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 somewhat limited. Traditional photosensitizers can be activated by short-wavelength light and have been successfully used in superficial cancers, such as skin and esophageal tumors. However, targeting deeper tumors remains challenging. Near-infrared II (NIR-II) refers to the spectral region with a wavelength of 1000-1700nm. The penetration depth of long-wavelength near-infrared (NIR) light is significantly higher than that of short-wavelength ultraviolet and visible light. Therefore, NIR light in NIR-II is considered to be the preferred light therapy for eliminating deep tumors because of its advantages such as higher maximum allowable exposure, deeper penetration, and low autofluorescence. Recently, a variety of conventional organic fluorescent dyes and AIE dyes that exhibit NIR-II fluorescence have been reported for use in bioimaging and biomedical applications. However, most of these dyes cannot effectively target mitochondria, probably due to their nonionic molecular structure and excessively high molecular weight.

[0006] Combination phototherapy, including PDT and PTT, has great potential to achieve local tumor destruction. However, their biomedical applications are also faced with challenges. Most PSs are based on the O2-dependent type II PDT mechanism, which involves energy transfer between excited-state PS and endogenous tumor O2, thereby generating cytotoxic O2. Unlike these O2-dependent type II PSs, type I PSs are independent of O2 and have better antitumor effects in the hypoxic tumor microenvironment.

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

[0008] The present invention overcomes the problems in the background technology and provides an AIE luminescent material in the second near-infrared region with mitochondrial targeting characteristics and a preparation method thereof.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

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

[0011]

[0012] A method for preparing a mitochondrial-targeted near-infrared zone II fluorescent dye with aggregation-induced emission properties comprises the following steps:

[0013] 5-(4-(di-p-tolylamino)phenyl)thiophene-2-carboxaldehyde, 1,1,2,3-tetramethyl-1H-benzo[e]indole hexafluorophosphate and 3-methylpiperidine were mixed, an appropriate amount of anhydrous ethanol was added, and the mixture was stirred at room temperature for 48 hours under a nitrogen atmosphere. After the solvent was evaporated under reduced pressure, dichloromethane / methanol was used as an eluent for elution, and the mixture was purified by silica gel column chromatography to obtain the target product;

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

[0015] A mitochondrial targeting red light dye BIH-TB, the molecular formula of which is: 27 H 24 F6NP, structural formula:

[0016]

[0017] A method for preparing a fluorescent dye BIH-TB having aggregation-induced emission properties, comprising mixing 5-phenylthiophene-2-carboxaldehyde, 1,1,2,3-tetramethyl-1H-benzo[e]indole hexafluorophosphate, 3-methylpiperidine and anhydrous ethanol, stirring at room temperature for 48 hours under a nitrogen atmosphere, evaporating the solvent under reduced pressure, and then purifying the mixture by silica gel column chromatography 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]indole hexafluorophosphate is 1:1.

[0019] The application of the mitochondria-targeted near-infrared luminescent dye in near-infrared fluorescence imaging.

[0020] The application of the mitochondrial-targeted near-infrared zone II luminescent dye with aggregation-induced luminescence properties in the preparation of mitochondrial-targeted reagents.

[0021] The application of the mitochondrial-targeted near-infrared zone II luminescent dye with aggregation-induced luminescence properties in the preparation of a preparation for phototherapy of tumors.

[0022] The application of the fluorescent dye BIH-TB in the preparation of mitochondrial targeting reagents.

[0023] The fluorescent dye BIH-TB is used in the preparation of a preparation for phototherapy of tumors.

[0024] Beneficial effects of the present invention:

[0025] 1. The near-infrared II (NIR-II) luminescent dye BIH-TA of the present invention has both mitochondrial targeting properties and aggregation-induced emission (AIE) properties, and has the following advantages compared with traditional photosensitizers:

[0026] (1) Good photostability, outstanding mitochondrial targeting, clear ROS generation mechanism, excellent photothermal performance, and large Stoke's shift.

[0027] (2) The near-infrared zone II aggregation-induced luminescent dye of the present invention has good mitochondrial targeting ability and near-infrared bio-imaging ability. Targeting mitochondria enables it to effectively destroy tumor mitochondria and thus kill tumor cells. The aggregation-induced luminescence property distinguishes it from traditional aggregation-quenched luminescent materials and has better bio-imaging effects. The near-infrared characteristics enable it to have good tissue penetration ability and bio-imaging effects. It has broad application prospects in near-infrared fluorescence imaging, preparation of mitochondrial targeting agents, and photodynamic therapy agents.

[0028] (3) The BIH-TA of the present invention exhibits strong NIR-II fluorescence, has typical AIE characteristics and significant mitochondrial targeting properties, and can be effectively applied to the combined PDT and PTT treatment of breast tumors, achieving good anti-tumor effects. Importantly, the synergistic induction of apoptosis and pyroptosis clarifies the molecular mechanism of combined phototherapy. These positive results open up the potential application of multifunctional NIR-IIAIE materials in combined phototherapy of breast tumors.

[0029] (4) The synthesis steps of the present invention are simple, which is conducive to commercial application and promotion.

[0030] The product was prepared by Knoevenagel condensation reaction at room temperature using ethanol as organic solvent and 3-methylpiperidine as alkaline catalyst. The chemical structure of the product was characterized by standard spectral analysis technology and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS), which confirmed that the chemical structure of the product was correct and the purity was high.

[0031] 2. The present invention also provides another red light dye BIH-TB with mitochondrial targeting properties, which emits bright fluorescence in the red and NIR regions, with an emission peak at 678nm and an absorption wavelength peak at 486nm. The preparation method of the material is simple and easy to implement. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Normalized fluorescence spectra of BIH-TA and BIH-TB;

[0033] Figure 2is the relationship between the relative fluorescence intensity (I / I0) and the composition of the tetrahydrofuran / water mixture of BIH-TA;

[0034] Figure 3 The temperature change of BIH-TA under different laser powers;

[0035] Figure 4 This is the generation of active oxygen in BIH-TA after laser irradiation;

[0036] Figure 5 This is the generation of hydroxyl radicals in the product after laser irradiation.

[0037] Figure 6 Colocalization imaging of BIH-TA stained U2-OS cells and commercial mitochondrial probes;

[0038] Figure 7 Comparison of the photostability of BIH-TA and commercial green mitochondrial probe in U2-OS cells obtained by continuous scanning under laser confocal microscopy;

[0039] Figure 8 Comparison of relative signal intensity of BIH-TA and commercial green mitochondrial probe in U2-OS cells under laser continuous scanning;

[0040] Fig. 9 Comparison of cytotoxicity of U2-OS cells after BIH-TA incubation under 660 nm laser irradiation and in the dark; Fig.10 Confocal fluorescence images of Calcein-AM and PI after incubation of U2-OS cells with BIH-TA in different experimental groups;

[0041] Fig.11 The graphs are the changes in relative tumor volume of mice in different experimental groups over time;

[0042] Fig.12 This is a graph showing the effect of BIH-TA on the tumor weight of mice;

[0043] Fig.13 These are NIR-II fluorescence images of mice at different monitoring time points after orthotopic tumor injection of BIH-TA;

[0044] Fig.14 for Fig.13 The mean fluorescence intensity changes over time;

[0045] Fig.15 for Fig.13 The signal-to-background ratio in the graph changes with time;

[0046] Fig.16In vitro NIR-II fluorescence images of different organs of mice 72 hours after injection of BIH-TA. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below with reference to 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 mitochondrial targeted near-infrared luminescent agent with aggregation-induced luminescence

[0049] Mitochondria-targeted near-infrared luminescent material (referred to as BIH-TA), with a molecular formula of C 41 H 37 F6N2PS, molecular weight is 734.79, structural formula is:

[0050]

[0051] The synthetic route is as follows:

[0052]

[0053] in,

[0054]

[0055] The preparation of BIH-TA is carried out by using ethanol as an organic solvent and 3-methylpiperidine as an alkaline catalyst, and obtaining the target product through Knoevenagel condensation reaction.

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

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

[0058] 1H NMR (500 MHz, dichloromethane-d2) 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 = 4.2 Hz, 1H), 7.76 (t, J = 7.2 Hz, 1H), 7.69-7.65 (m, 2H ),7.57(d,J=8.7Hz,2H),7.45(d,J=4.2Hz,1H),7.16(d,J=7.9Hz,4H),7.06(d,J=8.3Hz,4 H), 6.97 (d, J = 8.7Hz, 2H), 6.90 (d, J = 15.4Hz, 1H), 4.09 (s, 3H), 2.35 (s, 6H), 2.05 (s, 6H).

[0059] 13 C NMR (126 MHz, dichloromethane-d2) 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), so the subsequent solvent was changed to DMSO-d6 (dimethyl sulfoxide-d6). 13 C NMR(151MHz,DMSO-d6)δ180.95,154.01,149.29,144.32,143.68,139.80,139.50,138.10,137.38,133.80,132.93,130.80,130.3 4,130.01,128.33,127.31,126.86,126.73,125.51,125.01,124.38,123.04,120.03,113.02,109.00,53.20,34.23,25.39,20.45. 19 F NMR(470MHz,DMSO-d6)δ-69.70(d,J=709.7Hz), 31 P NMR(202MHz,DMSO-d6)δ-144.14(sept,J=711.0).FTMS(APCI source)m / z:[M-PF6]+ Calculate for C 41 H 37 N2S + ,589.2672;found,589.2672.

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

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

[0062] Mitochondrial targeted fluorescent dye BIH-TB (referred to as BIH-TB), with a molecular formula of C 27 H 24 F6NPS, molecular weight is 539.52, and its structural formula is:

[0063]

[0064] Preparation of BIH-TB compounds:

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

[0066] The chemical structure of the obtained product was characterized and the following data were obtained:

[0067] 1 H NMR(500MHz,DMSO-d6)δ8.75(d,J=16.0Hz,1H),8.44(d,J=8.4Hz,1H),8.29( d,J=8.9Hz,1H),8.23-8.20(m,2H),8.10(d,J=9.0Hz,1H),7.88-7.85(m,3H) ,7.82(ddd,J=8.3,6.8,1.4Hz,1H),7.73(t,J=7.5Hz,1H),7.55(t,J=7.5Hz, 2H),7.48(t,J=7.3Hz,1H),7.35(d,J=16.0Hz,1H),4.23(s,3H),2.02(s,6H). 13C NMR(151MHz,DMSO-d6)δ181.40(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 (470MHz, DMSO-d6) δ-73.23 (d, J=705.0Hz). 31 PNMR(202MHz,DMSO-d6)δ-144.14(sept,J=711.0).FTMS(APCI source)m / z:[M-PF6] + Calculate for C 27 H 24 NS + ,394.1624;found,394.1627.

[0068] Analysis shows that the synthetic method of the present invention obtains the target product BIH-TB.

[0069] Example 3 Use and performance of the mitochondrial targeting fluorescent dye of the present invention

[0070] (1) Normalized absorption and fluorescence spectroscopy detection

[0071] Normalized fluorescence spectra of the products of Examples 1 and 2 are shown in Figure 1 .

[0072] from Figure 1 It 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.

[0073] 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 low light scattering, low autofluorescence, and strong deep tissue penetration depth.

[0074] from Figure 1It 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.

[0075] (2) Fluorescence spectrum of the product

[0076] Figure 2 Figure 1 is a graph showing the relationship between the relative fluorescence intensity (I / I0) and the composition of a THF / water mixture of BTA. I0 = emission intensity in pure THF.

[0077] Example 1 Product BIH-TA at different water contents (f w ) in a tetrahydrofuran / water mixture. Figure 2 , where the concentration of BIH-TA solution is 0.1 mg / mL, the excitation wavelength is 585 nm, and the volume content of water in the tetrahydrofuran / water mixture is expressed as f w (vol%).

[0078] BIH-TA emits weak light in a solution of pure tetrahydrofuran. At the same concentration, in a tetrahydrofuran / water mixture, as the water content increases (10-60%), the solution fluorescence intensity decreases. As the water content further increases (70-90%), the fluorescence intensity of the solution increases significantly. When the water content reaches 90%, the fluorescence of the solution reaches a maximum value. This is because with the addition of the poor solvent water, the aggregation of BIH-TA molecules leads to enhanced fluorescence, indicating that BIH-TA has excellent aggregation-induced emission properties.

[0079] (3) Temperature changes of the product under different laser powers

[0080] Figure 3 The temperature change of BIH-TA under laser irradiation of different powers at a concentration of 200 μM and a wavelength of 660 nm.

[0081] It can be seen that when BIH-TA with a concentration of 200 μM is irradiated with lasers of different powers, its temperature increases with the increase of power, indicating that BIH-TA has excellent photothermal properties.

[0082] (4) Active oxygen generation of the product after laser irradiation

[0083] Using DCFH-DA (2',7'-dichlorofluorescein diacetate) as an indicator, the generation of reactive oxygen species after 660nm laser irradiation is shown in Figure 4 .

[0084] DCFH-DA reacts with active oxygen to emit fluorescence. When the solution without BIH-TA is irradiated with 660nm laser, the fluorescence intensity hardly changes. However, when the solution is irradiated with 660nm laser (1W / cm 2) The fluorescence intensity of the DCFH-DA solution containing BIH-TA increased significantly, indicating that BIH-TA produced a large amount of reactive oxygen species under 660nm laser irradiation. The increase in reactive oxygen species in tumor cells helps kill tumor cells.

[0085] Using HPF (hydroxyphenylfluorescein) as an indicator, the generation of hydroxyl radicals after 660nm laser irradiation is shown in Figure 5 .

[0086] HPF reacts with hydroxyl radicals to emit fluorescence. When the solution without BIH-TA is irradiated with 660nm laser, the fluorescence intensity hardly changes. However, when the solution is irradiated with 660nm laser (1W / cm 2 ) The fluorescence intensity of the HPF solution containing BIH-TA increased significantly, indicating that BIH-TA produced a large number of hydroxyl radicals under 660nm laser irradiation. This shows that BIH-TA can produce reactive oxygen species through the Type I pathway.

[0087] (5) Co-localization imaging of BIH-TA stained U2-OS cells of the present invention and commercial mitochondrial probes, see Figure 6 , where A: BIH-TA; B: commercial green mitochondrial probe; C: overlap; D: colocalization coefficient.

[0088] U2-OS cells were stained with a commercial green mitochondrial probe and BIH-TA for 30 min at 37°C and colocalized for imaging as follows:

[0089] BIH-TA at a concentration of 1 μM and a commercial green mitochondrial probe at a concentration of 1 μM were incubated with U2-OS cells for 30 minutes, respectively. Colocalization analysis was performed after imaging under a confocal microscope, and it was found that the colocalization coefficient (Pearson coefficient) was as high as 0.91, indicating that BIH-TA can target the mitochondria of cells.

[0090] (6) Comparison of photostability data of BIH-TA and commercial green mitochondrial probe in U2-OS cells.

[0091] Figure 7 Comparison of the photostability images of BIH-TA and commercial green mitochondrial probe in U2-OS cells scanned continuously by laser confocal microscopy.

[0092] from Figure 7 It can be seen that compared with commercial mitochondrial materials, BIH-TA has better photostability.

[0093] Comparison of relative signal intensities of BIH-TA and commercial green mitochondrial probe in U2-OS cells under laser continuous scanning, see Figure 8 .

[0094] BIH-TA (1 μM) and commercial green mitochondrial probe (1 μM) were incubated with U2-OS cells for 30 minutes, respectively, and scanned 90 times using a laser of the same power. The results showed that the relative signal intensity of BIH-TA decreased less, while the relative signal intensity of the commercial green mitochondrial probe decreased significantly, indicating that BIH-TA has good photostability.

[0095] (7) Effects of different concentrations of BIH-TA on U2-OS cell toxicity under laser irradiation

[0096] Comparison of cytotoxicity of U2-OS cells after BIH-TA incubation under 660 nm laser irradiation and in the dark, see Fig. 9 .

[0097] U2-OS cells were plated in a 96-well plate, with 10,000 cells per well. After the cells adhered to the wall, 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 and incubated for 12 hours. 2 ) were irradiated for 30 minutes, and after waiting for 12 hours, the cytotoxicity was tested by MTT method.

[0098] Under dark conditions, BIH-TA had little cytotoxicity within the tested concentration range, whereas after irradiation with a 660 nm laser, the cytotoxicity increased with increasing concentrations of BIH-TA, indicating that BIH-TA had excellent phototoxicity.

[0099] (8) Confocal fluorescence images of Calcein-AM and PI after incubation of U2-OS cells with BIH-TA

[0100] U2-OS cells were plated in confocal culture dishes, and when the confluence reached 80%, they were divided into four treatment 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).

[0101] The cells were then stained with Calcein-AM and PI and photographed using a confocal microscope. If the cells are alive, they emit green fluorescence; if the cells are dead, they emit red fluorescence. The results showed that only the BIH-TA+laser group was red, indicating that a large number of tumor cells in this group were killed, and the cells in the other three groups were active, indicating that BIH-TA has excellent phototoxicity.

[0102] Fig.10Confocal fluorescence images of Calcein-AM and PI after U2-OS cells were incubated 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. Scale bar: 100 μm.

[0103] (9) Animal experiment on tumor growth by BIH-TA

[0104] The subcutaneous breast cancer tumor model of Balb / c mice was established using 4T1 tumor cells. The experimental design included randomly dividing the 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.

[0105] (I) The PBS group received only PBS injection, and (II) the group received PBS injection and laser treatment. (III) The group received only BIH-TA treatment, and (IV) the group received BIH-TA treatment and laser treatment once, with a laser power of 0.5 W cm, a wavelength of 660 nm, and laser irradiation for 10 minutes. The body weight, tumor weight, and tumor volume of the mice were evaluated every two days for 12 days.

[0106] During the entire treatment period, the weight of mice in all groups increased slightly and remained basically the same. The tumor size of mice in groups (I), (II), and (III) increased significantly, increasing by 13, 14, and 12 times, respectively, on day 12. In contrast, the tumor volume of group (IV) with BIH-TA+laser remained basically unchanged, stabilizing at 220 mm 3 The weight of the tumor was significantly reduced, indicating that the tumor growth in this group was significantly inhibited. Fig.11 , Fig.12 .

[0107] (10) Tumor fluorescence imaging capability of BIH-TA

[0108] To investigate the tumor fluorescence imaging capability of BIH-TA, BIH-TA solution was injected into orthotopic 4T1 tumor-bearing mice, and then whole-body NIR-II fluorescence images were collected over time ( Fig.13 ). Intense NIR-II fluorescence was observed immediately after injection of BIH-TA, with a high signal-to-background ratio (SBR) of about 26. 72 h after injection, the emission intensity in the tumor area decreased to about 30% of the initial value ( Fig.14 ). The quantitative value of SBR 72h after injection followed a similar trend, decreasing from 26 to 7 ( Fig.15 ).

[0109] Ex vivo NIR-II fluorescence images of excised tumors and different organs in 4T1 xenograft mice 72 h after injection of BIH-TA ( Fig.16 ), further showed that BIH-TA was mainly present in the tumor site and partially in the liver and spleen, which may be related to its continuous metabolism in mice.

Claims

1. A mitochondrial-targeted near-infrared region II fluorescent dye with aggregation-induced emission properties, characterized in that: The molecular formula of the mitochondrial targeted near-infrared fluorescent dye is: 41 H 37 F6N2PS, the structural formula is 2. A method for preparing a mitochondrial-targeted near-infrared region II fluorescent dye with aggregation-induced emission properties, characterized in that: The following steps are involved: 5-(4-(di-p-tolylamino)phenyl)thiophene-2-carboxaldehyde, 1,1,2,3-tetramethyl-1H-benzo[e]indole hexafluorophosphate and 3-methylpiperidine were mixed, an appropriate amount of anhydrous ethanol was added, and the mixture was stirred at room temperature for 48 hours under a nitrogen atmosphere. After the solvent was evaporated under reduced pressure, dichloromethane / methanol was used as an eluent for elution, and the mixture was purified by silica gel column chromatography to obtain the target product; The molar ratio of 5-(4-(di-p-tolylamino)phenyl)thiophene-2-carboxaldehyde to 1,1,2,3-tetramethyl-1H-benzo[e]indole hexafluorophosphate is 1:

1.

3. A mitochondrial-targeted red dye BIH-TB, characterized in that: The molecular formula of the mitochondrial targeted red dye BIH-TB is: 27 H 24 F6NP, structural formula:

4. A method for preparing the fluorescent dye BIH-TB having aggregation-induced emission properties according to claim 3, characterized in that: 5-phenylthiophene-2-carboxaldehyde, 1,1,2,3-tetramethyl-1H-benzo[e]indole hexafluorophosphate, 3-methylpiperidine and anhydrous ethanol were mixed, stirred at room temperature for 48 hours under a nitrogen atmosphere, the solvent was evaporated under reduced pressure, and then purified by silica gel column chromatography using dichloromethane / methanol as an eluent to obtain a solid powder; The molar ratio of 5-phenylthiophene-2-carboxaldehyde and 1,1,2,3-tetramethyl-1H-benzo[e]indole hexafluorophosphate is 1:

1.

5. Use of the mitochondrial-targeted near-infrared luminescent dye with aggregation-induced emission properties as claimed in claim 1 in near-infrared fluorescence imaging.

6. Use of the mitochondrial-targeted near-infrared zone II luminescent dye with aggregation-induced emission properties as claimed in claim 1 in the preparation of mitochondrial-targeted reagents.

7. Use of the mitochondrial-targeted near-infrared zone II luminescent dye with aggregation-induced emission properties as claimed in claim 1 in the preparation of a preparation for phototherapy of tumors.

8. Use of the fluorescent dye BIH-TB according to claim 3 in preparing a mitochondrial targeting reagent.

9. Use of the fluorescent dye BIH-TB according to claim 3 in the preparation of a preparation for phototherapy of tumors.

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