Compound with aggregation-induced emission and application thereof in fluorescence imaging and photodynamic therapy

Abstract

The invention provides a compound with aggregation-induced emission and application thereof in fluorescence imaging and photodynamic therapy, and relates to the technical field of aggregation-induced emission materials. The compound 2TPA-PIMe with aggregation-induced emission (AIE) performance is constructed by taking triphenylamine as an electron donor group and alkylated phosphoindole as an electron acceptor group. As a photosensitizer, the compound 2TPA-PIMe provided by the invention has the advantages of chemical stability, light stability and low toxicity, and has the capability of efficiently generating I-type and II-type ROS (reactive oxygen species) under the irradiation of low-power white light. The compound can rapidly penetrate through cell membranes, target mitochondria and endoplasmic reticulum, and achieves an efficient anti-tumor effect through a dual ROS activation mode and dual organelle targeting capability. Meanwhile, the 2TPA-PIMe has a remarkable tumor retention characteristic, and conditions are provided for application of the 2TPA-PIMe in biological fluorescence imaging. The 2TPA-PIMe with the aggregation-induced emission characteristic, provided by the invention, has a huge application prospect in biological fluorescence imaging and photodynamic therapy.

Description

Technical Field

[0001] The present invention relates to the technical field of aggregation-induced luminescence materials, and in particular to a compound with aggregation-induced luminescence and applications thereof in fluorescence imaging and photodynamic therapy. Background Art

[0002] Cancer has become one of the most important health problems worldwide, and in China, cancer has become the leading cause of death. The most terrifying thing about cancer is its metastatic properties. It is reported that more than 90% of cancer patients die from metastasis. For example, osteosarcoma is the most common primary malignant bone tumor and the second leading cause of tumor-related death in adolescents. It is prone to lung metastasis and brain metastasis in the early stage. In addition, bones are also an important target for metastasis. Most malignant tumors will metastasize to the bone during their development. For example, up to 65%-80% of breast cancer and prostate cancer will metastasize to the bone during their development, and the bone metastasis rate of thyroid cancer, lung cancer and kidney cancer is as high as 35%-42%. Tumors metastasized to the bone usually cause corresponding motor dysfunction and neurological symptoms in the affected tissues, as well as pathological fractures and severe pain. At present, surgical resection, radiotherapy and chemotherapy are the three main methods for clinical treatment of tumors. However, these treatments all have certain limitations. Surgery usually cannot completely remove tumor boundary tissues and potential micro-lesions, increasing the risk of tumor recurrence and secondary metastasis, and is powerless against widely metastatic tumor lesions. Surgical treatment may also cause damage to joints and bone support structures, leading to postoperative loss of mobility and reduced quality of life. Chemotherapy drugs used clinically lack targeting capabilities and have serious side effects at therapeutic doses, such as bone marrow suppression and nephrotoxicity, as well as drug resistance after long-term use. The problem with radiotherapy (abbreviated as radiotherapy) is that most tumors are insensitive to radiation, especially in areas such as the spinal cord and nervous system, where the risk of radiotherapy is higher. Therefore, there is an urgent need to research and develop new and effective treatments to reduce or alleviate the suffering of patients with this disease.

[0003] Photodynamic therapy (PDT) has been developed into an effective tumor treatment method due to its excellent spatiotemporal precision and minimally invasive characteristics. PDT releases cytotoxic reactive oxygen species (ROS) through type I (electron transfer) or type II (energy transfer) reactions. ROS rapidly interact with biological substances and exert anti-tumor effects by inducing cell apoptosis or necrosis, vascular damage, and immune response. To date, various types of photosensitizers (PSs) have been reported, including inorganic nanomaterials, metal-organic frameworks, semiconductor polymer nanoparticles, and pure organic molecules. However, the potential cytotoxicity and complex synthesis of inorganic and metallic materials have largely limited their application. In contrast, pure organic molecules have irreplaceable advantages such as good biocompatibility, structural certainty, and functional modifiability. In addition, traditional organic photosensitizers have aggregation-caused quenching (ACQ) characteristics, weak photostability, and narrow Stokes shift, which limit their clinical application. Different from the ACQ characteristics of conventional organic photosensitizers, the aggregation-induced emission (AIE) characteristics endow PS with more stable fluorescence properties, achieving excellent properties such as good high-brightness fluorescence imaging, high stability, high ROS production efficiency, good biocompatibility, tumor targeting, and efficient tumor cell killing effect.

[0004] Based on this, the present invention aims to provide a new compound with aggregation-induced emission properties and study the application prospects of the compound in fluorescence imaging and photodynamic therapy. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of the shortcomings of the prior art, the present invention designs and synthesizes a compound (2TPA-PIMe) having a DAD structure, using triphenylamines (TPA) as an electron donor (Electron-donating, D) group and alkylated phosphindole (PIMe) as an electron acceptor (Electron-acceptor, A) group. The compound 2TPA-PIMe provided by the present invention has the advantages of chemical stability, photostability and low toxicity as a photosensitizer, as well as the ability to efficiently generate type I and type II ROS under low-power white light irradiation. The compound can quickly pass through the cell membrane, target mitochondria and endoplasmic reticulum, and achieves efficient anti-tumor effects through dual ROS activation modes and dual organelle targeting capabilities.

[0007] (II) Technical solution

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0009] In a first aspect, the present invention provides a compound having aggregation-induced emission, the structural formula of the compound being as shown in formula (I):

[0010] The compound is referred to as 2TPA-PIMe.

[0011] In a second aspect, the present invention provides a method for preparing the above-mentioned compound having aggregation-induced emission, wherein the compound is prepared according to the following steps:

[0012] (1) 2TPA-PI0) and 4-16 mmol trichlorosilane were mixed in 30-120 mL toluene at a molar ratio of 1-2:2-5, and stirred at room temperature for 2 h under nitrogen; then, 5-20 mL of saturated NaOH aqueous solution was added at 0°C, and then heated to room temperature, and the reaction was continued for more than 2 h; then, the solvent was evaporated under reduced pressure, and petroleum ether / dichloromethane (5:1 v / v) was used as the eluent, and the intermediate compound 2TPA-PI was purified by silica gel column chromatography to obtain;

[0013] (2) 2TPA-PI and methyl trifluoromethanesulfonate were mixed in 20-80 mL of dichloromethane at a molar ratio of 1-2:4-6, and stirred under nitrogen at room temperature for 12 h; then the solvent was removed, and the mixture was purified by silica gel column chromatography using dichloromethane / ethyl acetate as eluent to obtain the compound 2TPA-PIMe.

[0014] Furthermore, 2TPA-PIO was synthesized based on public literature, and the public literature information is: https: / / doi.org / 10.1039 / c6tc05591e ..

[0015] Furthermore, the chemical formula of 2TPA-PIO is shown in formula (II):

[0016]

[0017] Furthermore, the chemical formula of the intermediate compound 2TPA-PI is shown in formula (III):

[0018]

[0019] In a third aspect, the present invention provides the use of the above-mentioned compound 2TPA-PIMe in the preparation of photodynamic therapy drugs.

[0020] In a fourth aspect, the present invention provides the use of the above-mentioned compound 2TPA-PIMe in bioluminescence imaging, wherein the bioluminescence imaging is cell imaging and / or in vivo imaging and / or slice imaging.

[0021] (III) Beneficial effects

[0022] The present invention uses triphenylamines (TPA) as an electron-donating (D) group and alkylated phosphindole (PIMe) as an electron-acceptor (A) group to construct a compound 2TPA-PIMe with aggregation-induced emission (AIE) properties. And for the first time, the application of the compound 2TPA-PIMe in bioluminescent imaging and photodynamic therapy is discussed.

[0023] The 2TPA-PIMe provided by the present invention has strong absorption characteristics in the visible light region, and its absorption spectrum in the DMSO solution is located at 435nm, and the fluorescence emission spectrum is located at 650nm, showing a significant Stokes shift, which is conducive to improving the sensitivity of biological imaging. At the same time, in a mixed solution of DMSO and water, the fluorescence intensity of 2TPA-PIMe decreases first and then increases with the increase of water content, showing AIE characteristics; and dynamic light scattering and transmission electron microscopy confirm that it can form spherical nano-aggregates in aqueous solution. Under white light irradiation, 2TPA-PIMe can effectively generate reactive oxygen species (ROS), including hydroxyl radicals and singlet oxygen. It shows that the 2TPA-PIMe provided by the present invention has the potential for efficient photodynamic therapy (PDT).

[0024] We further confirmed that 2TPA-PIMe can be effectively taken up by MG-63 cells and mainly localized in mitochondria and endoplasmic reticulum, and has good in vitro anti-tumor effect. The 2TPA-PIMe provided by the present invention can be used as a good mitochondrial and endoplasmic reticulum organelle dual-targeting probe to achieve dual-targeted killing of tumor cells. In addition, laser confocal microscopy imaging confirmed that 2TPA-PIMe has the ability to efficiently produce reactive oxygen species (ROS) under white light irradiation. The MTT method confirmed that 2TPA-PIMe has good photodynamic efficiency and good cytotoxicity under illumination conditions; at the same time, according to the safety assessment results, 2TPA-PIMe did not cause hepatotoxicity and nephrotoxicity in vivo, and no abnormalities were found in blood and histological examinations, confirming that 2TPA-PIMe has good biocompatibility.

[0025] The results of in vivo animal experiments show that the 2TPA-PIMe provided by the present invention has significant tumor retention properties, which provides conditions for its application in bioluminescence imaging. At the same time, PDT mediated by 2TPA-PIMe shows excellent anti-tumor effects.

[0026] The 2TPA-PIMe with aggregation-induced emission characteristics provided by the present invention has great application prospects in bioluminescence imaging and photodynamic therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the synthetic route of compound 2TPA-PIMe.

[0028] Figure 2 The photophysical properties, particle size and morphology of the compound 2TPA-PIMe: (A) Absorption spectrum of 2TPA-PIMe in DMSO (10 μM); (B) Emission spectrum of 2TPA-PIMe in DMSO (10 μM); (C) Emission spectrum of 2TPA-PIMe in DMSO with different f w s(10μM) in DMSO / water mixture; (D) Emission spectrum of 2TPA-PIMe in f w =99 vol% dynamic light scattering (DLS) graph; (E) transmission electron microscope image of 2TPA-PIMe.

[0029] Figure 3 The results of ROS detection in the aqueous solution of compound 2TPA-PIMe are shown in Figure 2. (A) DCFH (10 μM) was used as an indicator to detect ROS under white light irradiation (20 mW cm -2 ) under white light irradiation (20 mW cm-1). I0 and I are the fluorescence intensities of the indicator before and after irradiation, respectively; (B) DHR123 (5 μM) was used as an indicator to detect the fluorescence intensity of the indicator under white light irradiation (20 mW cm-1). -2 ) under white light irradiation (20 mW cm -2 ) 1 O2. A0 and A are the absorbance of ABDA (10 μM) before and after irradiation, respectively.

[0030] Figure 4Results of cellular uptake, targeting, ROS generation and photodynamic effect of compound 2TPA-PIMe: (A) Uptake of 2TPA-PIMe by MG-63 cells; (B) Relative fluorescence quantification after treatment for different time periods. Scale bar: 10 μm; (C) Co-localization images of MG-63 cells with 2TPA-PIMe (1 μM) and Mito-Tracker Red, ER-Tracker Red, and Lyso-Tracker Green. Scale bar: 5 μm; (D) Cell viability in the dark or under white light after treatment of MG-63 cells with different concentrations of 2TPA-PIMe; (E) Live / dead staining detection of MG-63 cells; (F) Total ROS generation of 2TPA-PIMe in MG-63 cells after treatment with PBS + light (PBS + L), 2TPA-PIMe, and 2TPA-PIMe + light (2TPA-PIMe + L) (white light: 40 mW cm -2 10min). Scale bar: 50μm.

[0031] Figure 5 Results of in vivo photodynamic therapy of compound 2TPA-PIMe: (A) Schematic diagram of construction of subcutaneous 4T1 tumor model in Balb / C mice and in vivo anti-tumor treatment; (B) Fluorescence images of mouse tumors at different time periods after intratumoral injection of 2TPA-PIMe; (C) Photos of 4T1 tumor resection in mice in different treatment groups (n=5); (D) Growth curve of subcutaneous 4T1 tumor in mice in different treatment groups; (E) Weight of subcutaneous 4T1 tumor in mice in different treatment groups; (F) Changes in body weight of subcutaneous 4T1 tumor-bearing mice in different treatment groups. Data are expressed as mean±SD, n=5. (p<0.05, *; P<0.01, **; P<0.001, ***; P<0.0001, ****; not significant, ns); (G) H&E staining images, immunohistochemistry and immunofluorescence images (CleavedCaspase-3, Ki-67 and TUNEL) of tumor tissues in different groups after treatment. Scale bar: 20μm.

[0032] Figure 6 Routine blood test and blood biochemistry analysis of mice in each group after 10 days of treatment: (A) white blood cells (WBC), (B) red blood cells (RBC), (C) albumin (ALB), (D) alanine aminotransferase (ALT), (E) total bilirubin (TBIL), (F) alkaline phosphatase (ALP), (G) urea nitrogen (BUN), (H) creatinine (CREA) and (I) uric acid (UA).

[0033] Figure 7 H&E stained sections of the main organs of mice in each group after 10 days of treatment. Scale bar: 100 μm.

[0034] Figure 8 The compound 2TPA-PIMe is used for in vivo imaging; (A) is a single-photon CLSM image of a zebrafish embryo after staining with 2TPA-PIMe for 1 hour (10 μM); (B) is a 3D reconstruction of a local magnified image of the eyeball. The excitation wavelength laser is 488 nm, and the emission wavelength is 506-734 nm.

[0035] Fig. 9 The compound 2TPA-PIMe is used for cell imaging, live imaging and slice imaging; (A) is a two-photon CLSM image of 4T1 cells after being stained with 2TPA-PIMe for 30 minutes (1μM); (B) is a two-photon CLSM image of mouse kidney frozen sections treated with 2TPA-PIMe for 1 hour (5μM); (CD) is a two-photon CLSM image and a local magnified image of the zebrafish embryo eyeball after being stained with 2TPA-PIMe for 1 hour (10μM). The excitation wavelength of the two-photon laser excitation is 850nm. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Example 1

[0038] Preparation of a compound 2TPA-PIMe with aggregation-induced emission

[0039] 1 Preparation of compound 2TPA-PIMe

[0040] (1) 2TPA-PIO was synthesized according to the public literature (Public literature: Zhuang, Z.; Bu, F.; Luo, W.; Peng, H.; Chen, S.; Hu, R.; Qin, A.; Zhao, Z.; Tang, B. Z. Steric, conjugation and electronic impacts on the photoluminescence and electroluminescence properties of luminogens based on phosphindole oxide. Journal of Materials Chemistry C 2017, 5(7), 1836-1842, https: / / doi.org / 10.1039 / c6tc05591e .)

[0041] (2) Synthesis of intermediate compound (2TPA-PI): 2TPA-PIO (1.42 g, 2 mmol) and trichlorosilane (0.27 g, 4 mmol) were mixed in toluene (30 mL) and stirred at room temperature under nitrogen for 2 h. Saturated aqueous NaOH solution (5 mL) was added to the mixture at 0°C and the mixture was heated to room temperature. After extraction, the solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography using petroleum ether / dichloromethane (5:1 v / v) as the eluent to obtain a green solid (1.25 g, 90%).

[0042] The results of hydrogen and carbon NMR spectra of 2TPA-PI are as follows: 1 H NMR (400MHz, DMSO), δ (TMS, ppm): 7.67 (dd, J = 7.5, 4.7Hz, 1H), 7.47–7.15 (m, 18H), 7.13–6.98 (m, 12H), 6.94 (d, J = 8.1Hz, 4H), 6.71 (d, J = 8.7Hz, 2H). 13 C NMR (100MHz, DMSO), δ (TMS, ppm): 147.46, 147.35 (d, J = 5.0Hz), 147.11, 146.49, 1 44.67(d,J=5.0Hz),144.14(d,J=9.0Hz),142.50,133.86,133.69,133.28(d,J=1 9.0Hz),131.11,130.87,130.77,130.55,130.19,130.02,130.00,129.59,129.3 8(d,J=8.0Hz),128.96,126.58,124.97,124.53,123.99,123.87,123.68,121.81.

[0043] (3) Synthesis of 2TPA-PIMe: 2TPA-PI (1.25 g, 2 mmol) and methyl trifluoromethanesulfonate (1.31 g, 8 mmol) were mixed in 20 mL of dichloromethane and stirred under nitrogen at room temperature for 12 h. After evaporation of the solvent, the mixture was purified by silica gel column chromatography using dichloromethane / ethyl acetate as the eluent. A red solid of 2TPA-PIMe was obtained with a yield of 75% (1.07 g).

[0044] The results of hydrogen and carbon nuclear magnetic resonance spectra and high-resolution mass spectrometry of 2TPA-PIMe are as follows: 1H NMR (500MHz, CD2Cl2), δ (TMS, ppm): 8.05 (dd, J=10.9, 6.8Hz, 1H), 7.89–7.77 (m, 4H), 7.71– 7.60(m,4H),7.36–7.24(m,10H),7.18–7.06(m,13H),6.80(s,4H),2.65(d,J=14.0Hz,3H). 13 C NMR (125MHz, CD2Cl2), δ (TMS, ppm): 13 C NMR(126MHz,Methylene Chloride-d2)δ156.82(d,J=20.0Hz),150.90,150.17,147.89(d,J=35.0Hz),137.01,136.98,136.95, 133.43(d,J=11.25Hz),132.93(d,J=10.0Hz),132.08(d,J=13.75Hz),132.06,131.97,131.56,131.11( d,J=6.25Hz),130.88(d,J=2.50Hz),127.80(d,J=11.25Hz),127.02,126.90,125.78,125.63,124.28,1 24.17,123.15(d,J=11.25Hz),122.56,122.03,120.94,118.67,118.01,6.82(d,J=26.25Hz).HRMS:m / z calcd for C51H40N2P+:711.2924[M+]; found:711.2948.

[0045] The synthesis steps of TPA-PIMe are as follows Figure 1 shown.

[0046] 2 Characterization of compound 2TPA-PIMe

[0047] 2.1 UV-visible absorption and photoluminescence spectra of compound 2TPA-PIMe

[0048] 2TPA-PIMe was dissolved in DMSO to a concentration of 10 μM, 2 mL was taken into a quartz cuvette, and placed in a UV spectrophotometer and a fluorescence spectrometer for testing, and its absorption and emission spectra were recorded.

[0049] 2.2 AIE property test of compound 2TPA-PIMe

[0050] 2TPA-PIMe was dissolved in DMSO / water mixed solvents with different water contents (10 μM), excited with its maximum absorption wavelength, and its emission spectrum was recorded.

[0051] 2.3 Morphology of 2TPA-PIMe nanoparticles detected by transmission electron microscopy

[0052] (1) Dissolve 2TPA-PIMe in an aqueous solution containing 1 vol% DMSO to a certain concentration

[0053] (2) Take 10 μL of solution and drop it on the copper grid

[0054] (3) Leave to dry overnight

[0055] (4) Transmission electron microscopy imaging

[0056] 2.4 Dynamic light scattering detection of nanoparticle size of compound 2TPA-PIMe

[0057] 2TPA-PIMe was dissolved in an aqueous solution containing 1 vol% DMSO at 10 μM and measured at room temperature using a nanoparticle size potential analyzer (Zetasizer Nano-S90).

[0058] 2.5 Characterization results of compound 2TPA-PIMe

[0059] According to the absorption and photoluminescence spectra of 2TPA-PIMe ( Figure 2 AB), the maximum absorption peak of 2TPA-PIMe in DMSO is 435nm, and the fluorescence emission peak in DMSO is 650nm. 2TPA-PIMe exhibits a large Stokes shift (nearly 220nm), which may be caused by its multi-rotor. A large Stokes shift helps reduce the interference of background fluorescence and improve the sensitivity of biological imaging.

[0060] Meanwhile, since 2TPA-PIMe dissolves well in DMSO but is almost insoluble in water, its AIE properties in a mixture of DMSO and water were studied by changing the composition of water (fws). Figure 2 As shown in Figure C, with the addition of water, the fluorescence intensity of 2TPA-PIMe first weakens, which may be due to the twisted intramolecular charge transfer (TICT) effect, and then enhances with the further addition of water (fw>60%), which may be due to the dominance of the AIE effect.

[0061] In addition, dynamic light scattering (DLS) analysis showed that nanoaggregates were formed at fw = 99 vol%, and the average hydrated particle size (Dh) of 2TPA-PIMe was 72.25 nm ( Figure 2 D). Transmission electron microscopy (TEM) imaging further confirmed the formation of nanoparticles ( Figure 2 E).

[0062] 3 Detection of ROS generation by compound 2TPA-PIMe in aqueous solution

[0063] 3.1 Detection method

[0064] (1) Detection of total ROS in aqueous solution: DCFH was used as an indicator to detect the total ROS under white light irradiation (20 mW cm -2 ) ROS generation of 2TPA-PIMe in aqueous solution. First, DCFH-DA is activated to DCFH by conventional methods. Then DCFH, 2TPA-PIMe and Ce6 are dissolved in PBS so that the concentration of DCFH is 10 μM and the concentration of 2TPA-PIMe and Ce6 is 1 μM. Using 504nm as the excitation light source wavelength, the fluorescence signal of DCFH is observed in the wavelength range of 510 to 560nm. The change of fluorescence intensity is recorded at 525nm as an indication of the ROS generation rate.

[0065] (2) In aqueous solution 1 O2 detection: ABDA was used as an indicator and detected under white light irradiation (20 mW cm -2 ) in aqueous solution 1 O2 generation. ABDA, 2TPA-PIMe and Ce6 were dissolved in PBS so that the concentration of ABDA was 10 μM and the concentration of 2TPA-PIMe and Ce6 was 1 μM. Using 480 nm as the excitation light wavelength, the absorption spectrum of ABDA was measured in the wavelength range of 330 to 450 nm. The decrease in the absorption spectrum of ABDA was recorded at 380 nm to indicate 1 The rate of generation of O2.

[0066] (3) Detection of H2O2 in aqueous solution: DHR123 was used as an indicator to detect the H2O2 concentration under white light irradiation (20 mW cm -2) in aqueous solution. DHR123, 2TPA-PIMe and Ce6 were dissolved in PBS so that the concentration of DHR123 was 5 μM and the concentration of 2TPA-PIMe and Ce6 was 1 μM. The absorption spectrum of HPF was monitored in the range of 500-550 nm with an excitation wavelength of 480 nm. The change in fluorescence intensity was recorded at 525 nm to indicate the rate of H2O2 generation.

[0067] 3.2 Results

[0068] Since 2TPA-PIMe has strong absorption characteristics in the visible light region, its ability and efficiency to generate ROS in aqueous solution were evaluated under white light irradiation, using the commonly used clinical photosensitizer chlorin e6 (Ce6) and PBS as controls. First, DCFH was used as an indicator to detect the total ROS generation efficiency. DCFH can be excited by ROS and emit fluorescence at 525nm. Figure 3 As shown in A, with 20 mW cm -2 After white light irradiation of a DCFH aqueous solution containing 1 μM 2TPA-PIMe, the DCFH fluorescence intensity increased rapidly, and eventually increased by about 420 times within 5 minutes, showing a very high ROS generation efficiency. Under the same white light irradiation, the fluorescence intensity of the PBS group hardly increased, while that of the Ce6 group increased by less than 100 times. According to the mechanism of ROS generation, PDT can be divided into type I and type II. The former generates free radicals through electron transfer, while the latter generates singlet oxygen (singletoxygen, 1 Compared with type II PDT, type I PDT is less dependent on oxygen and is more suitable for the hypoxic microenvironment of tumors. Due to disproportionation and Harber-Weiss / Fenton reactions, superoxide anion radicals (O2 ·- ), hydroxyl radicals (·OH) and hydrogen peroxide (H2O2), which effectively kill tumor cells.

[0069] To further investigate the nature of ROS generated by 2TPA-PIMe, we used dihydrorhodamine 123 (HDR123) and 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) to detect H2O2 and 1 O2 and. Figure 3 As shown in B, in the presence of 2TPA-PIMe (1 μM), the -2After irradiation with white light for 5 minutes, the emission intensity of DHR123 increased by 7.14 times, indicating the production of H2O2. However, the fluorescence intensity of the PBS group and the Ce6 group remained almost unchanged after irradiation under the same conditions. At the same time, under the same conditions, the absorption intensity of the ABDA solution containing 2TPA-PIMe decreased by about 61%. Similarly, the absorption intensity of the PBS group remained almost unchanged, and the absorption of ABDA in the Ce6 group decreased by only 11% ( Figure 3 C) These results indicate that 2TPA-PIMe can efficiently generate type I and type II ROS, making it an ideal candidate photosensitizer for efficient PDT of tumors.

[0070] Example 2

[0071] Cellular uptake and organelle targeting of compound 2TPA-PIMe

[0072] After MG-63 cells were co-incubated with 2TPA-PIMe (1 μM) for different time periods (0.5, 1, 2, 4, 10, and 24 h), laser confocal microscopy was used for imaging. Fluorescence signals were observed in the cells at 0.5 h, indicating that it has good cell penetration ability. Moreover, as the incubation time increases, the fluorescence signals in the cells gradually increase in a time-dependent manner ( Figure 4 AB).

[0073] The subcellular localization of 2TPA-PIMe in MG-63 cells was further investigated by co-incubation with commercial dyes (Mito-Tracker Red, ER-Tracker Red, and Lyso-Tracker Green) and laser confocal imaging. Figure 4 As shown in Figure C, 2TPA-PIMe is mainly localized in the mitochondria and endoplasmic reticulum of MG-63 cells, with colocalization coefficients as high as 90% and 84%, respectively, as reflected by the overlap of red and green fluorescence signals. This indicates that 2TPA-PIMe is a good mitochondrial and endoplasmic reticulum dual-targeted organelle probe, making it possible to dual-target tumor cell killing.

[0074] Example 3

[0075] Study on the photodynamic effect of compound 2TPA-PIMe in vitro

[0076] Using DCFH-DA as a total ROS indicator, the ability of 2TPA-PIMe to generate ROS in MG-63 cells under white light irradiation was detected by laser confocal imaging. Figure 4 As shown in E, the cells were incubated with 1 μM 2TPA-PIMe for 4 h and then illuminated with 40 mW cm -2After 5 min of white light irradiation, the intracellular green fluorescence signal was significantly enhanced (2TPA-PIMe+L), while almost no fluorescence signal was generated in the PBS+L and 2TPA-PIMe groups, indicating that it is an efficient ROS generator.

[0077] The dark or phototoxicity of 2TPA-PIMe on cells was evaluated by MTT assay to further study the reliability and photodynamic efficiency of its biological applications. MG-63 cells were incubated with different concentrations of 2TPA-PIMe for 4 h and then treated with 40 mW cm -2 The cells were irradiated with white light for 10 min and then incubated in the dark for 20 h. The cells in the dark group were incubated in the dark for 24 h. Figure 4 As shown in D, 2TPA-PIMe showed negligible cytotoxicity in the absence of light and exhibited good biocompatibility. As the concentration of 2TPA-PIMe increased, cell viability decreased significantly, with more than 50% of cells losing activity at 1 μM and more than 90% of cells dying at 2 μM, indicating that 2TPA-PIMe has good photodynamic efficiency.

[0078] To further observe the PDT effect of 2TPA-PIMe, the Calcein-AM / PI co-staining method was used, with Calcein-AM (green) marking live cells and PI (red) marking dead cells, followed by laser confocal imaging. Figure 4 As shown in F, cells in the PBS+L or 2TPA-PIMe group (1 μM) showed only green fluorescence, indicating that there was no obvious cell membrane rupture and the cell viability was good. -2 ) cells showed a large area of ​​red fluorescence in the field of view, indicating cell membrane rupture and cell death. These results confirmed that 2TPA-PIMe has a good in vitro anti-tumor effect.

[0079] Example 4

[0080] In vivo imaging and photodynamic therapy studies of compound 2TPA-PIMe

[0081] We selected the mouse breast cancer cell line 4T1 as a cell model and constructed a subcutaneous tumor-bearing mouse model to evaluate the photodynamic efficiency of 2TPA-PIMe in vivo. Schematic diagram of the construction of the Balb / C mouse subcutaneous 4T1 tumor model and in vivo anti-tumor treatment is shown in the figure. Figure 5 As shown in A.

[0082] First, the imaging effect of 2TPA-PIMe in tumors was evaluated. 3In vivo experiments were started at 1:10 pm. In the animal imaging evaluation part, 0.5 h after the in situ injection of 2TPA-PIMe (5 mg / kg, 50 μL) into the tumor, a bright fluorescent signal appeared in the tumor site, and the fluorescent signal remained stable until 72 h, without diffusing into the normal tissue area. This indicates that 2TPA-PIMe has significant tumor retention properties and shows great potential in tumor imaging ( Figure 5 B).

[0083] Subsequently, we evaluated the antitumor effect of 2TPA-PIMe-mediated PDT in vivo. The mice were randomly divided into 4 groups: PBS group, PBS plus light group (PBS+L), 2TPA-PIMe group, and 2TPA-PIMe plus light group (2TPA-PIMe+L). When the tumor grew to about 150 mm 3 At 14:1, 2TPA-PIMe (5 mg / kg, 50 μL) or PBS (50 μL) was injected into the tumor. In order to obtain better accumulation of 2TPA-PIMe in the tumor, treatment was performed 24 h after injection. The PBS+L group and the 2TPA-PIMe+L group were illuminated with white light (200 mW cm -2 ) irradiated the tumor site for 30 min. Although 2TPA-PIMe can remain in the tumor for a long time, in order to obtain a better therapeutic effect, intratumoral injection was performed every 3 days. The treatment was repeated 3 times during the entire experiment. The mice were weighed and the tumor volume was measured every 2 days. After the experiment, the tumor was dissected and weighed. Figure 5 As shown in CD, the tumors in the PBS group, PBS+L group, and 2TPA-PIMe group grew rapidly, reaching nearly 1000 mm in just 11 days. 3 The tumors in the 2TPA-PIMe+L group were significantly inhibited and even significantly reduced, with the volume reduced by 5 times compared with the control group (P<0.01). Figure 5 As shown in Figure E, the tumor mass in the PBS group also increased 9-fold compared with the 2TPA-PIMe+L group (P<0.001). This indicates that the 2TPA-PIMe-mediated PDT process has a significant anti-tumor effect. Importantly, the body weights of mice in each group were close to each other, and no unacceptable weight loss was observed in any mouse during the treatment period (P>0.05), which means that 2TPA-PIMe has good biocompatibility ( Figure 5 F).

[0084] H&E staining showed that the tumors in the 2TPA-PIMe group had obvious cavitation, a large number of cell nuclei missing and obvious nuclear changes, indicating that PDT successfully destroyed the tumor tissue. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and cleaved-caspase-3 staining further verified the severe apoptosis of tumor cells induced by 2TPA-PIMe under white light irradiation. At the same time, through Ki-67 immunohistochemistry analysis, the tumor proliferation in the 2TPA-PIMe+L group was significantly inhibited, while the tumor cells in other groups were densely arranged and had extremely strong proliferation activity ( Figure 5 G).

[0085] Example 5

[0086] In vivo safety evaluation of compound 2TPA-PIMe

[0087] After 11 days of intervention, the mice in each group in Example 4 were killed and blood was collected for biochemical analysis. Figure 6 As shown in CI, biochemical indices such as alanine aminotransferase (ALT), total bilirubin (TBIL), alkaline phosphatase (ALP), alanine albumin (ALB), urea nitrogen (BUN), creatinine (CREA), and uric acid (UA) in each treatment group were within the normal range, and the differences were not statistically significant, indicating that the application of 2TPA-PIMe had no hepatotoxicity and nephrotoxicity.

[0088] It is worth noting that routine blood tests showed that after 2TPA-PIMe+L treatment, the red blood cell indicators of mice were normal, and all white blood cell indicators were close to normal levels, while in the other three groups, these white blood cell indicators were seriously abnormal due to severe tumor progression ( Figure 6 AB).

[0089] The main organs (heart, liver, spleen, lung, and kidney) of each group of mice were taken for histological analysis. Figure 7 As shown in the figure, no obvious damage or inflammatory changes were found in the organs stained with H&E. These results confirm that 2TPA-PIMe has good biocompatibility in vivo and has good application prospects in biology.

[0090] Example 6

[0091] The compound 2TPA-PIMe provided by the present invention can also be used for biological fluorescence imaging, including cell imaging, living imaging and slice imaging. Figure 8 A is a single-photon CLSM image of zebrafish embryos after staining with 2TPA-PIMe for 1 hour (10 μM); Figure 8 B is a 3D reconstruction of a magnified image of the eyeball. The excitation wavelength laser is 488nm and the emission wavelength is 506-734nm.

[0092] Fig. 9 A is a two-photon CLSM image of 4T1 cells after staining with 2TPA-PIMe for 30 minutes (1 μM); Fig. 9 B is a two-photon CLSM image of a mouse kidney cryosection treated with 2TPA-PIMe for 1 hour (5 μM); Fig. 9 CD are two-photon CLSM images and local magnified images of zebrafish embryo eyeballs stained with 2TPA-PIMe for 1 hour (10 μM). The excitation wavelength of two-photon laser excitation is 850 nm.

[0093] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A compound having aggregation-induced emission, the structural formula of the compound being as shown in formula (I): The compound is referred to as 2TPA-PIMe.

2. The method for preparing a compound having aggregation-induced emission according to claim 1, characterized in that: The compound is prepared according to the following steps: (1) 2TPA-PIO and 4-16 mmol trichlorosilane were mixed in 30-120 mL toluene at a molar ratio of 1-2:2-5, and stirred at room temperature for 2 h under nitrogen; then, 5-20 mL of saturated NaOH aqueous solution was added at 0°C, and then heated to room temperature and the reaction was continued for more than 2 h; then, the solvent was evaporated under reduced pressure, and petroleum ether / dichloromethane (5:1 v / v) was used as the eluent, and the intermediate compound 2TPA-PI was obtained by silica gel column chromatography purification; (2) 2TPA-PI and methyl trifluoromethanesulfonate were mixed in 20-80 mL of dichloromethane at a molar ratio of 1-2:4-6, and stirred under nitrogen at room temperature for 12 h; then the solvent was removed, and the mixture was purified by silica gel column chromatography using dichloromethane / ethyl acetate as eluent to obtain the compound 2TPA-PIMe.

3. The method for preparing a compound having aggregation-induced emission according to claim 2, characterized in that: The chemical formula of 2TPA-PIO is shown in formula (II):

4. The method for preparing a compound having aggregation-induced emission according to claim 2, characterized in that: The chemical formula of the intermediate compound 2TPA-PI is shown in formula (III):

5. Use of the compound 2TPA-PIMe as claimed in claim 1 in the preparation of photodynamic therapy drugs.

6. Use of the compound 2TPA-PIMe as claimed in claim 1 in bioluminescence imaging, wherein the bioluminescence imaging is cell imaging and / or in vivo imaging and / or slice imaging.

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