AIE-SPECT dual-modality imaging agent and its preparation method and application

Abstract

The present invention discloses an AIE-SPECT dual-modality imaging agent and its preparation method and application. It is based on a polymer-modified organic small molecule structure, adds a radionuclide with a SPECT imaging effect, forms nanoparticles through self-assembly, and chelates radionuclide ions to synthesize an AIE nanomaterial with both SPECT and fluorescence dual-modality imaging characteristics. The nanomaterial can produce near-infrared second-zone fluorescence under light irradiation in a specific wavelength range, and can produce a photothermal effect, so the nanomaterial can be used for tumor fluorescence imaging and photothermal therapy; at the same time, since the radionuclide can release beta rays, the nanomaterial can be used for tumor radiotherapy. The nanomaterial is uniformly dispersed in an aqueous solution, with an average particle size of 9.5nm, and can passively target tumors through the enhanced penetration and retention (EPR) effect, so it can be used as a tumor imaging agent and therapeutic drug.

Description

Technical Field

[0001] The present invention belongs to the field of tumor imaging and anti-tumor treatment, and specifically relates to an AIE (aggregation-induced emission)-SPECT (single photon emission computed tomography) dual-modality imaging agent and a preparation method and application thereof. The imaging agent can be used for tumor imaging, photothermal therapy and radiotherapy. Background Art

[0002] Cancer has become one of the biggest health risks worldwide. Although a variety of technologies and methods have been used for clinical tumor diagnosis and treatment, the heterogeneity and wide individual differences of tumors make it difficult for these methods to play a sufficient role and cannot benefit most cancer patients. In addition, diagnosis and treatment often require the use of different drugs, which increases the burden on patients. Therefore, the development of efficient integrated tumor diagnosis and treatment drugs is of great significance for accurate diagnosis and treatment of tumors and personalized treatment.

[0003] Aggregation-induced emission (AIE) molecules are a new type of fluorescent material that has emerged in recent years. Their fluorescence enhancement properties under aggregation conditions show unique application advantages. This type of material has stronger resistance to photobleaching and shows higher stability than traditional fluorescent materials. It can track disease progression stably and for a long time. The increase in fluorescence wavelength can enhance the tissue penetration of fluorescence and improve the spatial resolution of imaging. The emission wavelength of AIE molecules has gradually extended from visible light (400-700nm) to the near-infrared region II (1000-1700nm). Therefore, near-infrared region II AIE molecules have more important biological application potential. In addition, through structural optimization design, AIE materials with therapeutic properties can also be developed and used for tumor fluorescence imaging and treatment research. Among them, the photothermal effect of AIE molecules can be used to increase the local temperature of tumor tissue, thereby killing tumor cells, achieving targeted tumor clearance, and reducing the side effects of chemotherapy drugs on the human body.

[0004] The development of molecular imaging technology has provided an important tool for in vivo visualization analysis of tumors, which is of great significance for the early diagnosis of tumors. Although fluorescence imaging has a high spatial resolution, it has limited penetration into human tissues and is difficult to be used for whole-body tumor screening. Single-photon emission computed tomography (SPECT) is a technology that relies on the γ signal generated by the decay of radioactive nuclides to detect lesions. It has unlimited penetration into human tissues and can be used for whole-body imaging analysis; in addition, some radionuclides themselves can emit high-energy β rays, which can be used for tumor radiotherapy. The disadvantage is that its spatial resolution is limited, making it difficult to accurately locate local lesions and cannot be used for intraoperative imaging analysis. Therefore, combining fluorescence imaging with SPECT imaging can play a complementary role. SPECT imaging can be used for preliminary screening of lesions, and then fluorescence imaging can be used for local imaging and positioning to clearly outline the tumor boundary. Combining AIE molecules with photothermal effects with SPECT nuclides to develop tumor fluorescence-SPECT dual-modality imaging and therapeutic agents is of great significance to the development and transformation application of integrated tumor diagnosis and treatment technology.

[0005] At present, there is no report on AIE-SPECT imaging agents that have both fluorescence-SPECT dual-modality imaging and photothermal-radiotherapy characteristics. Therefore, the present invention provides an AIE-SPECT dual-modality imaging agent that can be used for tumor imaging and photothermal-radiotherapy, as well as a preparation method and application thereof, which has broad application prospects in the field of integrated tumor diagnosis and treatment research. Summary of the invention

[0006] The purpose of the present invention is to provide an AIE-SPECT dual-modality imaging agent that can be used for tumor imaging and photothermal-radiotherapy, as well as a preparation method and application thereof. The present invention uses AIE molecules as the core, water-soluble polymers as connecting molecules and chelating agents, and achieves radionuclide labeling by chelation, thereby preparing an imaging agent with fluorescence and SPECT dual imaging and photothermal-radiotherapy characteristics.

[0007] The imaging agent molecule of the present invention is denoted as 177 Lu-2TT-oC6B has the following structure:

[0008]

[0009] Among them, R is

[0010]

[0011] Its preparation method is:

[0012] (1) Synthesis of AIE molecules: At room temperature, the compound 2TT-oC6B-N 3Add DBCO-PEG2K-DOTA into anhydrous tetrahydrofuran to react and obtain compound 2TT-oC6B-DOTA;

[0013]

[0014] (2) Radionuclide labeling: The compound 2TT-oC6B-DOTA was dissolved in PBS (pH = 7.2-7.4) and 177 LuCl 3 Solution mixing reaction to obtain dual-modal imaging agent molecules 177 Lu-2TT-oC6B;

[0015] Wherein, the nuclide is 177 Lu, the nuclide ion is chelated with the DOTA group in the AIE molecule to achieve nuclide labeling.

[0016] The AIE-SPECT dual-modality imaging agent is used in the preparation of tumor fluorescence and SPECT imaging agents.

[0017] The application of the AIE-SPECT dual-modality imaging agent in the preparation of tumor photothermal therapy agents.

[0018] The AIE-SPECT dual-modality imaging agent is used in the preparation of tumor radiotherapy agents.

[0019] The use of the AIE-SPECT dual-modality imaging agent in the preparation of an integrated tumor diagnosis and treatment reagent.

[0020] The beneficial effects of the present invention are:

[0021] The present invention uses a covalent connection method to finally connect the AIE components and label the nuclides. Compared with the conventional coating method, this method can make the connection between the nuclides and the AIE molecules more stable, and the obtained polymer-modified AIE molecules also have a smaller size (the average particle size is basically below 10nm in the present invention) and stronger in vivo stability, which is more conducive to subsequent related applications; and compared with 2TT-oC6B, the AIE molecules obtained by the present invention not only have the AIE effect, but also have strong photothermal properties. Therefore, the present invention provides a multifunctional imaging agent that can be used for tumor near-infrared second-zone fluorescence imaging and SPECT imaging and photothermal-radiotherapy for the first time. This imaging agent can generate near-infrared second-zone fluorescence under specific wavelength light excitation, so that in vivo fluorescence imaging can be achieved. The labeled radionuclide can be used for SPECT imaging to achieve non-invasive tumor diagnosis. In addition, this imaging agent not only has a photothermal effect, but also can emit beta rays, and can be used for photothermal therapy and radiotherapy of local tumor tissues. Therefore, the developed AIE-SPECT dual-modality imaging agent can be used as both an imaging agent and a tumor therapeutic drug for dual-modality tumor imaging and photothermal-radiotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings.

[0023] Figure 1 is a synthetic route map of the AIE molecules described in the present invention;

[0024] Figure 2 The AIE molecule precursor 2TT-oC6B-N of the present invention 3 Synthesis route map;

[0025] Figure 3 This is a comparison diagram of infrared spectra of compounds 2TT-oC6B-DOTA and DBCO-PEG2K-DOTA;

[0026] Figure 4 Graphs showing the absorption and emission spectra of the AIE molecules in Example 1 of the present invention;

[0027] Figure 5 This is a particle size test diagram of the AIE molecule in Example 1 of the present invention;

[0028] Figure 6 The AIE molecule solutions with different concentrations in Example 1 of the present invention are at 1 W / cm 2 Temperature variation over time under laser irradiation;

[0029] Figure 7 The AIE molecule solution with a concentration of 60 μg / mL in Example 1 of the present invention is at 1 W / cm2 Heating-cooling cycle curve under laser irradiation;

[0030] Figure 8 This is an in vivo fluorescence imaging image of a tumor-bearing mouse after the imaging agent in Example 2 of the present invention is injected intravenously. DETAILED DESCRIPTION

[0031] Reagents and Materials

[0032] Tetrahydrofuran was purchased from Anage Reagent Company, and PBS was purchased from White Shark Biological Reagent Company.

[0033] Example 1

[0034] The synthesis route of the AIE molecule 2TT-oC6B-DOTA of the present invention is as follows Figure 1 As shown, the present invention is further described below in conjunction with a specific example:

[0035] Compound 2TT-oC6B-DOTA

[0036] At room temperature, 6.0 mg (29.1 mmol) of compound 2TT-oC6B-N 3 and 41.4 mg of DBCO-PEG2K-DOTA were added to 5 mL of anhydrous tetrahydrofuran, stirred at room temperature for 24 h, and the solvent was removed in vacuo to obtain a black solid product, namely 2TT-oC6B-DOTA 36.5 mg (yield: 77.0%).

[0037] Among them, compound 2TT-oC6B-N 3 The synthesis process of Figure 2 As shown, the preparation process is briefly described below.

[0038] Compound 2

[0039] Compound 1 (1.92 g, 5 mmol) was dissolved in 10 mL of anhydrous dichloromethane under nitrogen atmosphere. Boron tribromide (15 mL, 30 mmol, 2.0 M in DCM) was slowly added dropwise to the solution at 0 °C. After stirring at room temperature for 2 hours, the reaction was quenched by adding water at 0 °C. The organic phase was extracted with dichloromethane and brine and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation and dried in a vacuum oven to obtain compound 2.

[0040] Compound 4

[0041] Compound 2 (1.43 g, 4 mmol) and anhydrous potassium carbonate (3.32 g, 24 mmol) were dissolved in 30 mL of acetone under a nitrogen atmosphere, and compound 3 (3.71 mL, 24 mmol) was added dropwise to the reaction solution, and the mixture was stirred at 80°C for 12 hours. After cooling to room temperature, the mixture was filtered and washed with acetone, and the filtrate was removed by rotary evaporation to obtain a crude product, which was further separated and purified by silica gel column chromatography to obtain compound 4.

[0042] Compound 6

[0043] Under nitrogen atmosphere, compound 4 (1.36 g, 2 mmol), compound 5 (1.37 g, 3 mmol), tetrakis(triphenylphosphine)palladium (Pd(PPh 3 ) 4 (115.56 mg, 0.1 mmol) was dissolved in 30 mL of anhydrous toluene, and the mixture was refluxed for 24 h. After cooling to room temperature, the solvent was removed by rotary evaporation, and the crude product was separated and purified by silica gel column chromatography to obtain compound 6.

[0044] Compound 7

[0045] At -78 ° C and nitrogen atmosphere, n-butyl lithium (n-BuLi 0.46mL, 1.1mmol, 2.4M in hexane) was dropped into 20mL of THF solution of compound 6 (769.72mg, 1mmol) and stirred at -78 ° C for 1h. Then tributyltin chloride (1.1mL, 1.1mmol, 1.0M in THF) was added to the solution, and after stirring at room temperature for 12 hours, KF solution was added to quench the reaction. Extracted three times with n-hexane, the organic phase was dried over anhydrous sodium sulfate. After removing the solvent, compound 7 was obtained without further purification.

[0046] Compound 9

[0047] Under nitrogen atmosphere, compound 7 (847.02 mg, 0.8 mmol), compound 8 (70.40 mg, 0.2 mmol), tris dibenzylideneacetone dipalladium (Pd 2 (dba) 3 18.31mg, 0.02mmol), tri(o-methylphenyl)phosphine (P(o-tol) 3 48.70 mg, 0.16 mmol) was dissolved in 20 mL of anhydrous toluene. The mixture was refluxed for 24 h. After cooling to room temperature, the solvent was removed by rotary evaporation. The crude product was separated and purified by silica gel column chromatography to obtain compound 9.

[0048] Compound 2TT-oC6B-N 3

[0049] Under nitrogen atmosphere, compound 9 (173.0 mg, 0.1 mmol) and sodium azide (NaN 3 65.01 mg, 1 mmol) was dissolved in 20 mL of acetonitrile and refluxed for 24 h. After cooling to room temperature, the organic phase was extracted with ethyl acetate and brine, dried over anhydrous sodium sulfate, and then rotary evaporated to obtain a crude product, which was further separated and purified by neutral alumina column chromatography to obtain compound 2TT-oC6B-N 3 .

[0050] Example 2

[0051] Radionuclide labeling

[0052] To 0.5 mL of the AIE molecule 2TT-oC6B-DOTA in PBS buffer (pH = 7.2-7.4), 10 μL of the radionuclide was added. 177 The Lu solution was reacted at 37°C for 1 hour to complete the radionuclide labeling and obtain the AIE-SPECT dual-modality imaging agent.

[0053] Example 3

[0054] In vitro photothermal performance testing of AIE molecules

[0055] The AIE molecules prepared in Example 1 were diluted proportionally to solutions of different concentrations, respectively placed in different centrifuge tubes, and irradiated with a 750 nm laser. The temperature change of the solution was monitored in real time with a thermal imager, and recorded every 30 seconds.

[0056] The photothermal effect test results of AIE molecules are shown in Figure 6 As the irradiation time increases, the solution temperature gradually rises and tends to be stable. Increasing the concentration of the imaging agent can increase the temperature difference of the solution. At a concentration of 180 μg / mL, the temperature difference can reach more than 40°C, reflecting strong photothermal performance and having potential application value in tumor photothermal therapy.

[0057] Example 4

[0058] In vitro photothermal stability testing of AIE molecules

[0059] The AIE molecule prepared in Example 1 was diluted to 60 μg / mL and placed in a centrifuge tube. The solution was irradiated with a 750 nm laser and the temperature change of the solution was monitored in real time with a thermal imager, with recording every 30 seconds.

[0060] The results of the photothermal stability test of AIE molecules are shown in Figure 7As the irradiation time increases, the solution temperature gradually rises. After the irradiation stops, the solution temperature drops rapidly. This cycle repeats 5 times. It can be seen that the solution temperature does not change significantly when the heating is turned on and off, which reflects the strong photothermal stability of the developer, which can withstand long-term illumination while maintaining photothermal performance.

[0061] Example 5

[0062] Fluorescence imaging of tumor-bearing mice

[0063] The imaging device prepared in Example 2 was intravenously injected into tumor-bearing mice, and fluorescent images of the mice were captured at different times using an in vivo fluorescent imager.

[0064] The results of fluorescence imaging of tumors are shown in Figure 8 As time goes by, the fluorescence signal at the tumor site gradually increases, which shows that the imaging agent is continuously accumulated in the tumor tissue through blood circulation, and the contrast gradually increases, reflecting the imaging agent's efficient targeting and aggregation characteristics on the tumor and its excellent fluorescence imaging effect.

[0065] The imaging agent obtained by the present invention can be uniformly dispersed in an aqueous solution, with an average particle size of 9.5 nm, and it can be seen that the obtained product can generate near-infrared second-zone fluorescence under near-infrared light excitation, thereby realizing in vivo fluorescence imaging. The labeled radionuclide can be used for SPECT imaging, which can realize non-invasive tumor diagnosis. Thus, the present invention obtains an AIE-SPECT dual-modality imaging agent;

[0066] In addition, this imaging agent has an excellent and stable photothermal effect and can be used for photothermal therapy of local tumor tissues; at the same time, the radionuclide can generate beta rays for tumor radiotherapy. Therefore, the AIE-SPECT dual-modality imaging agent prepared by the present invention can be used as an imaging agent and a tumor therapeutic drug for dual-modality imaging and photothermal-radiotherapy of tumors. Moreover, the imaging agent can passively target tumors through the enhanced penetration and retention (EPR) effect and can be used as a tumor imaging agent and therapeutic drug.

[0067] The above description is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An AIE-SPECT dual-modality imaging agent, It is characterized in that The imaging agent molecule is denoted as 177 Lu-2TT-oC6B, the structural formula is as follows: ; Among them, R is 。 2. The method for preparing the AIE-SPECT dual-modality imaging agent according to claim 1, It is characterized in that The preparation method of the imaging agent molecule comprises: (1) Synthesis of AIE molecules: At room temperature, the compound 2TT-oC6B-N 3 Add DBCO-PEG2K-DOTA to anhydrous tetrahydrofuran and perform click chemistry reaction to obtain compound 2TT-oC6B-DOTA; ; Among them, R is ; (2) Radionuclide labeling: The compound 2TT-oC6B-DOTA was dissolved in PBS and 177 LuCl 3 Solution mixing reaction to obtain dual-modal imaging agent molecules 177 Lu-2TT-oC6B.

3. Use of the AIE-SPECT dual-modality imaging agent as claimed in claim 1 in the preparation of tumor fluorescence and SPECT imaging agents.

4. Use of the AIE-SPECT dual-modality imaging agent as claimed in claim 1 in the preparation of a tumor photothermal therapy agent.

5. Use of the AIE-SPECT dual-modality imaging agent as claimed in claim 1 in the preparation of tumor radiotherapy agents.

6. Use of the AIE-SPECT dual-modality imaging agent according to claim 1 in the preparation of an integrated tumor diagnosis and treatment reagent.

7. An AIE molecule with photothermal effect, It is characterized in that The AIE molecule is 2TT-oC6B-DOTA, and its structural formula is ； Among them, R is 。

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