Near-infrared second window emitting small molecule rare earth complex fluorescent probe and preparation method thereof

Abstract

The present application belongs to the technical field of biological materials, and particularly relates to a near-infrared second window emission small-molecule rare earth metal complex fluorescent probe and a preparation method thereof.The fluorescent probe comprises a complex formed by coordination of DOTA and a rare earth Ln(III) metal, a complex formed by coordination of DOTA-NHS ester and the rare earth Ln(III) metal, and a product obtained by reaction of Ln-DOTA-NHS and amino PEG folic acid.The present application changes the functional groups of a macrocyclic polyamine structure without affecting the coordination ability of the macrocyclic polyamine and the rare earth ion, and achieves the effect of targeting tumors after specific binding with receptors on the surface of tumors, and simultaneously realizes surgical resection under the guidance of a near-infrared window.According to the donor type of chemical modification of the small-molecule rare earth complex fluorescent probe, the molecular probe can specifically target various tumors, realize an antibody-antigen immune response, and realize gene expression through base pair pairing of a DNA sequence.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a small molecule rare earth complex fluorescent probe and its preparation method. Background Technology

[0002] Currently, common molecular imaging techniques such as X-rays, computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound imaging (US) are used to assist in surgery, but these are not tumor-specific and their effectiveness in surgical applications is generally not ideal. Fluorescence imaging, due to its advantages such as real-time operation, non-invasiveness, small sample requirements, and high resolution, has been widely used in life sciences and biotechnology fields, especially tumor-specific fluorescence imaging methods, which have significant application value. In recent years, researchers have focused on fluorescence detection and imaging in the first near-infrared window (700nm–900nm). However, due to the strong absorption and scattering of biological tissues in this wavelength range, the signal-to-noise ratio and tissue penetration depth are relatively low. Therefore, recent research has mainly focused on the second near-infrared window (1000nm–1700nm), where the absorption and scattering of biological tissues are weak, which can greatly improve image quality and penetration depth. Currently, some inorganic materials such as carbon nanotubes, quantum dots, rare earth nanoparticles, and organic dye molecules can achieve emission in the near-infrared second window region. However, their emission peaks are relatively broad, their metabolism is slow after entering the living organism, they have potential cytotoxicity, and they have poor solubility in water, which greatly limits their application value.

[0003] Small-molecule rare-earth metal complexes can also achieve emission in the near-infrared second window region, and possess advantages such as large Stokes shift, sharp-line emission spectrum, long fluorescence lifetime, high luminescence efficiency, low photobleaching, and low long-term toxicity, making them a promising fluorescent probe for deep tissue in vivo imaging. The unique electronic structure of rare-earth ions endows them with many special spectral properties. Because the ff transition of rare-earth ions is a forbidden transition, their own light absorption is very weak, and their molar extinction coefficient is very small. Therefore, their luminescence is mainly achieved through ligand sensitization. Most current rare-earth complexes are visible-light emitting rare-earth complexes sensitized by ultraviolet-visible light. Therefore, rare-earth complexes with high luminescence efficiency under near-infrared light excitation would be an ideal fluorescent probe for deep tissue in vivo imaging and image-guided surgical resection. By modifying the functional groups of the macrocyclic structure without affecting the coordination ability of macrocyclic polyamines with rare-earth ions, donors can be chemically modified to target tumors. After specific binding to receptors on the tumor surface, the tumor can be targeted, simultaneously achieving near-infrared window-guided surgical resection. Depending on the donor type used in the chemical modification of small-molecule rare-earth complex fluorescent probes, molecular probes can specifically target various tumors, enabling antibody-antigen immune responses and gene expression through DNA base pairing. They hold broad application prospects in clinical bioanalysis and disease detection. Summary of the Invention

[0004] The purpose of this invention is to provide a small molecule rare earth metal complex fluorescent probe with simple preparation process, good water solubility, high photostability, rapid renal clearance, and applicable to in vivo imaging and imaging-guided surgical resection in deep tissues with near-infrared emission through the second window, as well as its preparation method.

[0005] The near-infrared second window emission small molecule rare earth metal complex fluorescent probe provided by this invention is a complex formed by coordination of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) with a rare earth Ln(III) metal, denoted as Ln-DOTA, and its general structural formula is as follows:

[0006]

[0007] Furthermore, the near-infrared second window emission small molecule rare earth metal complex fluorescent probe provided by the present invention is a complex formed by coordination of the DOTA derivative DOTA-NHS ester and the rare earth Ln(III) metal, denoted as Ln-DOTA-NHS, and its general structural formula is as follows:

[0008]

[0009] Furthermore, the near-infrared second window emission small molecule rare earth metal complex fluorescent probe provided by the present invention is the product of the reaction between the above-mentioned rare earth complex fluorescent probe Ln-DOTA-NHS and aminoPEG folic acid (NH2-PEG-FA), denoted as Ln-DOTA-FA. It can be used as a near-infrared second window emission rare earth complex fluorescent probe targeting ovarian cancer, and its general structural formula is as follows:

[0010]

[0011] In this invention, the rare earth Ln(III) metal is a chloride, and the chloride is selected from: NdCl3·6H2O, ErCl3·6H2O, TmCl3·6H2O, or HoCl3·6H2O.

[0012] The method for preparing small molecule rare earth metal complex fluorescent probes proposed in this invention is as follows:

[0013] (1) Preparation of Ln-DOTA small molecule rare earth complex fluorescent probe.

[0014] Weigh 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and place it in a single-necked round-bottom flask, dissolving it in 1–3 ml of water. Then weigh rare earth chlorides (selected from NdCl3·6H2O, ErCl3·6H2O, TmCl3·6H2O, HoCl3·6H2O), dissolving them in 1–2 ml of water. Slowly add the chloride aqueous solution to the DOTA aqueous solution while stirring at room temperature. Adjust the pH to 6.0–6.5 with NaOH solution. React for 12–24 hours, then stop the reaction. Slowly add anhydrous diethyl ether or petroleum ether to precipitate the product, allow it to stand, filter, and obtain the corresponding solid powder, denoted as Ln-DOTA. The molar ratio of DOTA to rare earth chloride is 1:1 to 1:3, with 1:1 being the optimal condition.

[0015] (2) Preparation of Ln-DOTA-NHS small molecule neodymium complex fluorescent probe.

[0016] Weigh out the DOTA derivative DOTA-NHS ester (DOTA-NHS) and place it in a single-necked round-bottom flask, dissolving it in 1–3 ml of water. Then weigh out rare earth chlorides (selected from NdCl3·6H2O, ErCl3·6H2O, TmCl3·6H2O, HoCl3·6H2O), dissolving them in 1–2 ml of water. Slowly add the chloride aqueous solution dropwise to the DOTA-NHS ester aqueous solution while stirring at room temperature. Adjust the pH to 6.0–6.5 with NaOH solution. React for 12–24 hours, then stop the reaction. Slowly add anhydrous diethyl ether or petroleum ether to precipitate the product. Allow it to stand, filter, and obtain a white solid, denoted as Ln-DOTA-NHS. The molar ratio of DOTA-NHS ester to rare earth chloride is 1:1–1:3, with 1:1 being the optimal condition.

[0017] (3) Prepare Ln-DOTA-FA small molecule neodymium complex fluorescent probe.

[0018] Weigh the solid prepared in step (2), put it into a single-necked round-bottom flask, add N,N-diisopropylethylamine (DIPEA) and N,N-dimethylformamide (DMF), stir at room temperature, then weigh aminoPEG folic acid (NH2-PEG-FA), dissolve it in 1-2 ml of water, slowly add it dropwise to the Ln-DOTA-NHS solution, stir at room temperature for 12-24 hours, stop the reaction, concentrate the reaction solution, wash it several times with anhydrous diethyl ether or petroleum ether to obtain a yellow solid, which is the desired Ln-DOTA-FA small molecule neodymium complex fluorescent probe.

[0019] The mass ratio of N,N-diisopropylethylamine to N,N-dimethylformamide is 1:8 to 1:10, preferably 1:9.

[0020] In this invention, taking the small molecule rare earth complex fluorescent probe molecule Nd-DOTA as an example, it exhibits strong absorption at 740 nm and 802 nm in aqueous solution. Further excitation with an 808 nm laser reveals fluorescence emission peaks of rare earth neodymium ions at 1060 nm and 1330 nm. Figure 1 ).

[0021] In this invention, taking the small molecule rare earth complex fluorescent probe molecule Nd-DOTA as an example, its fluorescence lifetime at 1060 nm in aqueous solution is 5.37 μs.

[0022] In this invention, taking the small molecule rare earth complex fluorescent probe molecule Nd-DOTA as an example, the fluorescence quantum yield in aqueous solution is 0.3%.

[0023] In this invention, taking the small molecule rare earth complex fluorescent probe molecule Nd-DOTA as an example, under the excitation of an 808nm laser, its photostability remained unchanged for 2 hours in aqueous solution, PBS buffer solution, and mouse blood, respectively. Figure 2 ).

[0024] In this invention, taking the small molecule rare earth complex fluorescent probe molecule Nd-DOTA as an example, when the concentration is 500 μg / ml, the cell viability is still above 95%. Figure 3 ).

[0025] This invention modifies the functional groups of macrocyclic polyamines without affecting their coordination ability with rare earth ions, thereby chemically modifying donors that can target tumors. These donors then specifically bind to receptors on the tumor surface to achieve tumor targeting, while simultaneously enabling surgical resection guided by the near-infrared window.

[0026] Depending on the donor type used in the chemical modification of small-molecule rare-earth complex fluorescent probes, molecular probes can specifically target various tumors, enabling antibody-antigen immune responses and gene expression through base pairing in DNA sequences. They hold broad application prospects in clinical bioanalysis and disease detection.

[0027] The ligands that coordinate with rare earth metals in this invention are also applicable to the remaining five ligands of commercially available and clinically used MRI contrast agents: DOTA derivatives DO3A-butrol and HP-DO3A, DTPA and its diamide derivatives DTPA-BMA and DTPA-BMEA. Their corresponding structural formulas are as follows:

[0028]

[0029]

[0030] The donors used in this invention for chemical modification targeting include folic acid (FA), follicle-stimulating hormone (FSHβ), RGD peptide, matrix metalloproteinase (MMP), epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and some other antibodies and DNA sequences.

[0031] In this invention, the small molecule rare earth complex fluorescent probe, depending on the type of chemically modified donor, can specifically target various tumors to achieve antibody-antigen immune responses and gene expression through base pairing of DNA sequences. Attached Figure Description

[0032] Figure 1Absorption and fluorescence emission spectra of Nd-DOTA, a small molecule rare earth complex fluorescent probe molecule, excited at 808 nm near-infrared.

[0033] Figure 2 Photostable spectra of the small rare-earth complex fluorescent probe molecule Nd-DOTA in aqueous solution, PBS buffer, and nude mouse blood under 808 nm near-infrared excitation.

[0034] Figure 3 This is a cytotoxicity diagram of the small molecule rare earth complex fluorescent probe molecule Nd-DOTA.

[0035] Figure 4 The fluorescence emission spectra of the small molecule rare earth complex fluorescent probe molecule Nd-DOTA-NHS are obtained by 808nm near-infrared excitation.

[0036] Figure 5 The fluorescence emission spectra of the small molecule rare earth complex fluorescent probe molecule Nd-DOTA-FA are obtained by 808nm near-infrared excitation. Detailed Implementation

[0037] Example 1:

[0038] Preparation of the small molecule rare earth complex fluorescent probe molecule Nd-DOTA. The specific steps are as follows:

[0039] Weigh 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and dissolve it in 1–3 ml of water in a single-necked round-bottom flask. Then weigh NdCl3·6H2O and dissolve it in 1–2 ml of water. Slowly add the NdCl3 aqueous solution to the DOTA aqueous solution while stirring at room temperature. Adjust the pH to between 6.0 and 6.5 with NaOH solution. After reacting for 12–24 hours, stop the reaction. Slowly add anhydrous diethyl ether to precipitate. After standing, filter to obtain a light purple solid powder. The small molecule rare earth complex fluorescent probe molecule Nd-DOTA has strong absorption at 740 nm and 802 nm in aqueous solution. Further excitation with an 808 nm laser reveals fluorescence emission peaks of rare earth neodymium ions at 1060 nm and 1330 nm (see [link to relevant documentation]). Figure 1 ).

[0040] Example 2:

[0041] Preparation of the small molecule rare earth complex fluorescent probe molecule Nd-DOTA-NHS. The specific steps are as follows:

[0042] Weigh out DOTA derivative DOTA-NHS ester (DOTA-NHS) and dissolve it in 1-3 ml of water in a single-necked round-bottom flask. Then weigh out NdCl3·6H2O and dissolve it in 1-2 ml of water. Slowly add the NdCl3 aqueous solution to the DOTA-NHS aqueous solution while stirring at room temperature. Adjust the pH to 6.0-6.5 with NaOH solution. After reacting for 12-24 hours, stop the reaction. Slowly add anhydrous diethyl ether to precipitate. Let stand, filter, and obtain a white solid. The aqueous solution of the small molecule rare earth complex fluorescent probe molecule Nd-DOTA-NHS, when excited with an 808 nm laser, shows a strong emission peak of rare earth neodymium ions at 1060 nm and a weaker fluorescence emission peak at 1330 nm (see [link to relevant documentation]). Figure 4 ).

[0043] Example 3:

[0044] Preparation of the small molecule rare earth complex fluorescent probe molecule Nd-DOTA-FA. The specific steps are as follows:

[0045] Weigh out the solid Nd-DOTA-NHS and place it in a single-necked round-bottom flask. Add N,N-diisopropylethylamine (DIPEA) and N,N-dimethylformamide (DMF) (1 / 9). Stir at room temperature. Then weigh out aminoPEG-folic acid (NH2-PEG-FA), dissolve it in 1-2 ml of water, and slowly add it dropwise to the Nd-DOTA-NHS solution. Stir at room temperature for 12-24 hours, then stop the reaction. Concentrate the reaction solution and wash it several times with anhydrous diethyl ether to obtain a yellow solid. An aqueous solution of the small molecule rare earth complex fluorescent probe molecule Nd-DOTA-FA, when excited with an 808 nm laser, shows a strong emission peak of rare earth neodymium ions at 1060 nm and a weaker fluorescence emission peak at 1330 nm (see [link to relevant documentation]). Figure 5 ).

[0046] Example 4:

[0047] Treatment of the Nd-DOTA fluorescent probe molecule, a small molecule rare earth complex, for cytotoxicity assay. Cytotoxicity was assessed using CCK-8 reagent in CaVO3 cells. The specific steps are as follows:

[0048] CaVO3 cells (1×10) 4Cells were cultured in 96-well plates at 5% CO2 and 37°C for 24 hours. They were then further incubated for 24 hours under the same conditions with different concentrations of Nd-DOTA complex (0, 100, 200, 300, 400, 500 μg / mL, respectively). Finally, 10 μL of CCK-8 in PBS solution was added to each well, and the plates were incubated at 37°C for 4 hours. Cell viability was measured using a microplate reader. Measurements were based on absorbance at 450 nm. Cell viability can be calculated using the following formula:

[0049] Cell viability (%) = (average absorbance of experimental group / average absorbance of control group) × 100.

Claims

1. A near-infrared second window-emitting rare earth complex fluorescent probe, characterized in that, The complex is formed by coordination of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and its derivative DOTA-NHS with a rare earth metal Nd (III), and is denoted as Nd-DOTA-NHS, and has the following structural formula: ; Or the product of the reaction of the rare earth complex fluorescent probe Nd-DOTA-NHS with amino PEG folic acid, denoted as Nd-DOTA-FA, whose structural formula is as follows: .

2. The near-infrared second window-emitting rare earth complex fluorescent probe according to claim 1, characterized in that, The rare earth metal Nd(III) is the chloride NdCl36H2O.

3. A method for preparing a rare-earth complex fluorescent probe emitting through a near-infrared second window as described in claim 1, characterized in that: The specific steps for preparing Nd-DOTA-NHS are as follows: Weigh out the DOTA derivative DOTA-NHS and place it in a single-necked round-bottom flask, then dissolve it in 1-3 mL of water. Next, weigh out the rare earth chloride NdCl3·6H2O and dissolve it in 1-2 mL of water. Slowly add the chloride aqueous solution to the DOTA-NHS aqueous solution, stir at room temperature, adjust the pH with NaOH solution to between 6.0 and 6.5, and react for 12-24 hours. Stop the reaction, slowly add anhydrous diethyl ether or petroleum ether to precipitate, let stand, filter, and obtain a white solid, denoted as Nd-DOTA-NHS. The molar ratio of DOTA-NHS to rare earth chloride is 1:1 to 1:

3. The specific steps for preparing Nd-DOTA-FA are as follows: Weigh solid Nd-DOTA-NHS and place it in a single-necked round-bottom flask. Add N,N-diisopropylethylamine and N,N-dimethylformamide and stir at room temperature. Then weigh aminoPEG folic acid, dissolve it in 1-2 mL of water, and slowly add it dropwise to the Nd-DOTA-NHS solution. After stirring at room temperature for 12-24 hours, stop the reaction, concentrate the reaction solution, and wash it several times with anhydrous diethyl ether or petroleum ether to obtain a yellow solid, which is the desired Nd-DOTA-FA rare earth complex fluorescent probe. The mass ratio of N,N-diisopropylethylamine to N,N-dimethylformamide is 1:8 to 1:10.

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