Polyelectrolyte assembly based on aggregation-induced emission dye and preparation method and application thereof

Abstract

The application provides a kind of polyelectrolyte assembly based on aggregation-induced emission dye and a preparation method and application thereof.The dye is an aggregation-induced emission dye with bidentate ligand, and has the structure shown in formula I.The dye uses quinoline nitrile derivative as aggregation-induced emission unit, and uses m-phthalic acid and its derivatives as coordination unit, so that it has many advantages such as near-infrared fluorescence emission, large stokes shift, excellent metal coordination and good biocompatibility, and can construct stable polyelectrolyte assembly.The dye is assembled with cation-neutral block polymer and metal ion through coordination and electrostatic interaction to construct polyelectrolyte assembly.By coordinating with different functional metal ions, the polyelectrolyte assembly can realize multi-mode imaging integration.This kind of polyelectrolyte assembly based on aggregation-induced emission dye has been successfully used for in vivo real-time imaging of tumor mice.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of imaging, and particularly relates to a kind of polyelectrolyte assembly based on aggregation-induced emission dye and a preparation method thereof. BACKGROUND

[0002] In the prior art, it is very challenging to obtain multi-dimensional information of tumors by using detection means with high sensitivity and strong specificity. For single-mode imaging technology, resolution and sensitivity are usually a pair of opposite aspects that restrict each other. For example, fluorescence imaging (FL) mode can significantly improve the time resolution of imaging and is the preferred choice for surgical navigation, but it is difficult to obtain depth imaging information of tissues. Magnetic resonance imaging (MRI) can obtain tissue information without depth, but its imaging sensitivity is often limited. Positron emission tomography (PET) has high sensitivity and time resolution to some extent, but its spatial resolution is still insufficient. Therefore, how to develop a multi-mode imaging probe system, especially by combining the imaging advantages of different modes, to obtain information at the level of living body, tissue and even molecule with multi-scale and high resolution, is a key problem in the field of imaging.

[0003] Nanotechnology is an important means to realize in vivo diagnosis. It can not only integrate multi-mode imaging reagents into a single nanoparticle, but also significantly improve the pharmacokinetics and tumor targeting performance of the imaging reagents. The most common strategy is to physically encapsulate different functional reagents (such as fluorophores, magnetic resonance contrast reagents, drugs, etc.) into nanoparticles formed by amphiphilic block copolymers. However, this encapsulation strategy based on hydrophobic force may affect the imaging performance of its components. For example, the hydrophobic confinement environment in the interior of the nanoparticle hinders the interaction between the magnetic resonance contrast reagent and water protons, thereby greatly weakening the MRI signal. Therefore, how to synergistically improve the multi-mode imaging performance in the nanosystem while considering sensitivity and resolution is a difficult problem to be solved. SUMMARY

[0004] The purpose of the present application is to construct a kind of polyelectrolyte assembly based on aggregation-induced emission dye, to realize synergistically enhanced multi-mode imaging application. The dye is a bidentate ligand aggregation-induced emission dye. The dye uses quinoline nitrile derivative as the aggregation-induced emission unit, and pyridine-2,6-dicarboxylic acid as the coordination unit, so that it has near-infrared fluorescence emission, large stokes shift, excellent metal coordination and good biocompatibility, and many other advantages. The dye is assembled with cation-neutral block polymer and metal ions through coordination and electrostatic interaction, and can form stable polyelectrolyte assembly. By coordinating different metal ions (Gd 3+ , Eu 3+ , Ga 3+ , Mn 2+The polymeric micelles can be endowed with multiple imaging modes (MRI or PET) to realize multi-modal imaging integration. Notably, since the assembly driving force of the nanoparticles is the combination of coordination and ionic interaction, the nanoparticles have a "soft" and water-filled internal environment, thereby effectively improving the fluorescence signal and nuclear magnetic relaxation rate. The polymeric micelles realize high signal-to-noise ratio and specific tracking imaging in the live tumor of a mouse.

[0005] The object of the present application is achieved by the following scheme:

[0006] The polymeric micelle based on the aggregation-induced emission dye is an aggregation-induced emission dye with a bidentate ligand, and the structure is shown in formula I:

[0007]

[0008] In formula I, R1 is independently selected from any one of a cyano group (-CN), a carboxyl group (-COOH) or a group shown in formula II (wherein the curved mark is a substitution site, and the same applies below);

[0009]

[0010] X is independently selected from O, any one of a group shown in formula III or formula IV;

[0011]

[0012] R2 is independently selected from any one of a group shown in formula V or formula VI; Y is C or N; R3 is selected from any one of H, a methoxy group, an N,N-dimethylamino group or an N,N-diethylamino group;

[0013]

[0014] Another object of the present application is to provide a preparation method of the polymeric micelle based on the aggregation-induced emission dye, which is a preparation method of the aggregation-induced emission dye with a bidentate ligand. The steps include: Knoevenagel condensation reaction of a compound shown in formula VII with a corresponding aldehyde formula VIII or formula IX, and further defatting under alkaline conditions to obtain the target compound.

[0015]

[0016] wherein, X, Y and R1, R2 are defined as described above.

[0017] Further, R1 is a group shown in formula II;

[0018] X is O, any one of a group shown in formula III and formula IV;

[0019] R2 is any one of the groups shown in formula V and formula VI; Y is C or N; R3 is selected from any one of H, methoxy, N,N-dimethylamino or N,N-diethylamino;

[0020] In a further preferred technical solution, X is a group shown in formula IV; R2 is a group shown in formula VI; Y is C or N; R3 is selected from any one of H, methoxy, N,N-dimethylamino or N,N-diethylamino;

[0021] More preferably, Y is N; R3 is selected from any one of H, methoxy, N,N-dimethylamino or N,N-diethylamino;

[0022] More preferably, R3 is N,N-diethylamino;

[0023] The application provides a method for preparing a compound shown in formula I, and main specific steps of the method are as follows: a Knoevenagel condensation reaction is carried out on a compound shown in formula VII and corresponding aldehyde formula VIII or formula IX, and then defatting is carried out under alkaline conditions to obtain the target compound.

[0024]

[0025] Wherein, the definitions of X, Y and R1 and R2 are the same as described above.

[0026] The application further provides an application of a polyelectrolyte assembly based on an aggregation-induced emission dye, and the application is an application of the aggregation-induced emission dye with a bidentate ligand in construction of the polyelectrolyte assembly.

[0027] Further, the application is an application in multi-mode imaging, including fluorescence imaging, photothermal imaging, magnetic resonance imaging or positron emission tomography imaging.

[0028] The application provides a polyelectrolyte assembly based on an aggregation-induced emission dye, and realizes synergistically enhanced multi-mode imaging. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 . The dye I-2 is used for constructing the polyelectrolyte assembly together with the cation-neutral block polymer and the metal ion under the action of ultrasound.

[0030] Figure 2 . A particle characterization graph of the polyelectrolyte assembly TCM-Gd-PE under a cryogenic transmission electron microscope.

[0031] Figure 3 Figure 6. Size analysis curve of polyelectrolyte assembly TCM-Gd-PE within 7 days.

[0032] Figure 4 Figure 7. Size analysis curve of polyelectrolyte assembly constructed by dye I-2 and different metal ions (Eu 3+ , Tb 3+ , Zn 3+ , Mn 2+ ).

[0033] Figure 5 Figure 8. Comparison of magnetic relaxivity of polyelectrolyte assembly TCM-Gd-PE and commercial magnetic contrast agent Gd-DTPA.

[0034] Figure 6 Figure 9. Magnetic imaging of polyelectrolyte assembly TCM-Gd-PE and commercial magnetic contrast agent Gd-DTPA at different Gd 3+ concentrations.

[0035] Figure 7 Figure 10. In vivo magnetic imaging of polyelectrolyte assembly TCM-Gd-PE on tumor-bearing mice. DETAILED DESCRIPTION

[0036] In one preferred technical solution of the present application:

[0037] The present application is further described by the following examples, which are intended to better illustrate the present application and are not intended to limit the scope of the present application:

[0038] Example 1

[0039] (1) Synthesis of dye I-1:

[0040]

[0041] 4-(4-(4-Formylphenoxy)butoxy)-2,3-dihydropyridine-2,6-dicarboxylic acid diethyl ester (112 mg, 0.27 mmol), 3-(2,6-dimethyl-l-phenylpyridin-4(lH)-ylidene)-2-phenylprop-l-en-l,1,3-tricarbonitrile (100 mg, 0.27 mmol), piperidine 1 mL, acetic acid 0.5 mL and toluene 3 mL were added into a 10 mL single-neck flask, which was heated to 100 °C under argon protection for 24 h. After the reaction was completed, the solvent was removed by rotary evaporation, and the crude product was purified by silica gel column chromatography (dichloromethane). After rotary evaporation, an orange-red solid was obtained (148 mg, 0.13 mmol), with a yield of 47%.

[0042] 1H-NMR (400 MHz, CDC13, ppm): δ 1.33 (m, 12H, -CH3), 1.88 (s, 8H, -CH2-CH2-), 4.06 (s, 4H, -CH2-O), 4.28 (s, 4H, -CH2-O), 4.38 (t, 8H, -CH2-OCO), 6.11 (d, J = 15.6 Hz, 2H, alkene-H), 7.67-7.91 (m, 26H, Ph-H). Mass spectrometry (ESI-MS, m / z): [M+H] + calcd. for [C 69 H 99 N6O 12 +Na] + : 1191.4480; found: 1191.4475.

[0043]

[0044] The product from the previous step (110 mg, 0.094 mmol), KOH (420 mg, 7.52 mmol), tetrahydrofuran (20 mL) were added to a 50 mL single neck flask and stirred at room temperature for 6 h. After the reaction was completed, the solvent was removed by rotary evaporation and the crude product was purified by silica gel column chromatography (dichloromethane:methanol = 2:1) to give dye I-1 (95 mg, 0.08 mmol) as a red solid after rotary evaporation. Yield: 94%.

[0045] (2) Synthesis of dye I-2:

[0046]

[0047] Diethyl 4-(4-(5-(diethylamino)-2-formylphenoxy)butyloxy)pyridine-2,6- dicarboxylate (155 mg, 0.32 mmol), 3-(2,6-dimethyl-l-phenylpyridin-4(lH)-ylidene)-2- phenylprop-l-en-l,1,3-tricarbonitrile (120 mg, 0.32 mmol), piperidine 1 mL, acetic acid 0.5 mL and toluene 3 mL were added to a 10 mL single neck flask and heated to 100 °C for 24 h under argon protection. After the reaction was completed, the solvent was removed by rotary evaporation and the crude product was purified by silica gel column chromatography (dichloromethane) to give an orange red solid (245 mg, 0.19 mmol) after rotary evaporation. Yield: 58%.

[0048] 1H NMR (400 MHz, CDC13, ppm): δ 1.16 (t, J = 6.8 Hz, 12H, -CH3), 1.44 (t, J = 6.8 Hz, 12H, -CH3), 1.95-2.03 (m, 8H, -CH2-CH2-), 3.36 (q, J = 6.8 Hz, 8H, -CH2-N), 4.00 (t, J = 5.6 Hz, 4H, -CH2-O), 4.23 (t, J = 5.2 Hz, 4H, -CH2-O), 4.45 (q, J = 7.2 Hz, 8H, -CH2-OCO), 6.02 (s, 2H, Ph-H), 6.06 (d, J = 16.0 Hz, 2H, alkene-H), 6.16 (d, J = 8.4 Hz, 2H, Ph-H), 6.94 (d, J = 8.8 Hz, 2H, Ph-H), 7.35-7.37 (m, 2H, Ph-H), 7.44-7.65 (m, 12H, Ph-H and alkene-H), 7.75 (s, 4H, Ph-H). Mass spectrometry (ESI-MS, m / z): [M+Na] + calcd. for [C 77 H 82 N8O 12 +Na] + 1333.5950; found 1333.5939.

[0049]

[0050] The product of the previous step (40 mg, 0.03 mmol), KOH (67 mg, 1.2 mmol), tetrahydrofuran (20 mL) were added into a 50 mL two-necked flask and reacted at 25 °C for 48 h under argon protection. After the reaction was completed, the solvent was removed by rotary evaporation, and the crude product was purified by silica gel column chromatography (dichloromethane:methanol = 2:1). After rotary evaporation, dye I-2 (30 mg, 0.024 mmol) was obtained with a yield of 80%.

[0051] Example 2

[0052] Dye I-2 for constructing polyelectrolyte assemblies

[0053] As Figure 1 shown, the aggregation-induced emission dye I-2 (0.1 mM), the cationic-neutral block polymer (1.62 mM) and the metal ion Gd 3+ (0.067 mM) were added into a Tris buffer solution (10 mM, pH 7.4), and a polyelectrolyte assembly TCM-Gd-PE was prepared by ultrasonication at a frequency of 0.5 MHz for 2 min. As Figure 2As shown, TCM-Gd-PE has uniform and regular spherical structure by cryo-TEM characterization. As shown, Figure 3 As shown, TCM-Gd-PE can keep stable particle size within 7 days by dynamic light scattering analysis, and the diameter is about 67 nm. As shown, Figure 4 As shown, aggregation-induced emission dye I-2 can be coordinated and assembled with various metals, and these assemblies have similar particle sizes by dynamic light scattering analysis.

[0054] Example 3

[0055] In-vitro nuclear magnetic contrast of polyelectrolyte assembly TCM-Gd-PE

[0056] Relaxivity (r1) is an important indicator for evaluating the nuclear magnetic contrast of materials. We tested the relaxivity of polyelectrolyte assembly, and selected commercial magnetic contrast agent Gd-DTPA as a reference. As shown, Figure 5 As shown, the relaxivity of polyelectrolyte assembly TCM-Gd-PE is 38.06 mM -1 s -1 , which is about 8 times of that of commercial magnetic contrast agent Gd-DTPA (r1 = 4.88 mM -1 s -1 ). Further, we prepared TCM-Gd-PE and Gd-DTPA solutions with different Gd 3+ concentrations, and performed nuclear magnetic contrast under a magnetic field intensity of 3.0 T (instrument: MAGNETOM Prisma 3.0T). As shown, Figure 6 As shown, the pure water solution shows a low magnetic number; as the Gd 3+ concentration in the TCM-Gd-PE solution continuously increases, the magnetic number significantly increases. Moreover, compared with commercial Gd-DTPA, TCM-Gd-PE exhibits a stronger magnetic number, which is consistent with the results of the relaxivity test.

[0057] Example 4

[0058] In-vivo nuclear magnetic contrast of polyelectrolyte assembly TCM-Gd-PE in mice

[0059] All in-vivo experiments in the present application comply with the regulations for laboratory animal feeding and use, and are approved by the University Animal Feeding and Use Committee of East China University of Technology. The tumor-bearing nude mice used in the experiment were purchased from Shanghai Slek Animal Experiment Co., Ltd., and were fed in a sterile cage in a laminar flow hood in a sterile room, and were fed with high-pressure steam treated food and water.

[0060] The tumor-bearing nude mice were injected with TCM-Gd-PE (0.006 mmol kg -1 Gd 3+). After injection, MRI nuclear magnetic imaging was performed on the tumor-bearing nude mice using a MAGNETOM Prisma 3.0T. Before the imaging experiment, the nude mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution (45 mg / kg).

[0061] It can be seen from Figure 7 that after the polyelectrolyte assembly TCM-Gd-PE is injected into the tail vein, there is a significant increase in the magnetic number in the tumor of the mouse after 3 h, indicating that TCM-Gd-PE has good in vivo nuclear magnetic imaging capability and can be further applied to in vivo biological tests.

Claims

1. An aggregation-induced emission dye with bidentate ligand, having the structure of Formula I: Formula I wherein R1 is independently selected from any one of cyano (-CN), carboxyl (-COOH) and Formula II, wherein the curved arrow indicates the substitution site, and the same applies hereinafter; X is independently selected from Formula IV; R2 is independently selected from Formula V and Formula VI; Y is C or N; and R3 is selected from any one of H, methoxy, N,N-dimethylamino or N,N-diethylamino.

2. The dye of claim 1, wherein R1 is Formula II, X is Formula IV, R2 is Formula V, Y is C or N, and R3 is selected from any one of H, methoxy, N,N-dimethylamino or N,N-diethylamino.

3. The dye of claim 1, wherein R1 is Formula II, X is Formula IV, R2 is Formula V, Y is C, and R3 is selected from any one of H, methoxy, N,N-dimethylamino or N,N-diethylamino.

4. The dye of claim 1, wherein R1 is Formula II, X is Formula IV, R2 is Formula V, Y is N, and R3 is selected from any one of H, methoxy, N,N-dimethylamino or N,N-diethylamino.

2. The aggregation-induced emission dye with bidentate ligand according to claim 1, characterized in that, 5. The dye of claim 1, wherein R1 is Formula II, X is Formula IV, R2 is Formula V, Y is N, and R3 is N,N-diethylamino.

3. The aggregation-induced emission dye with bidentate ligand according to claim 1, wherein, 6. A method of preparing the dye of claim 1, comprising the steps of: subjecting a compound of Formula VII to Knoevenagel condensation reaction with a corresponding aldehyde of Formula VIII or Formula IX, and further de-esterification under basic conditions to obtain the target compound, wherein X, Y, R1 and R2 are as defined above.

4. The aggregation-induced emission dye with bidentate ligand according to claim 1, wherein, 7. Use of the dye of claim 1 in constructing polyelectrolyte assemblies.

5. The aggregation-induced emission dye with bidentate ligand according to claim 1, wherein, 8. Use of the dye of claim 1 in fluorescence imaging, photo-thermal imaging, magnetic resonance imaging or positron emission tomography imaging. ​ ​ wherein ​ ​ ​

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