Near-infrared two-region fluorescent conjugated polymer with anti-aggregation quenching effect and preparation method and application thereof

Abstract

The application discloses a near-infrared two-region fluorescent conjugated polymer with an anti-aggregation quenching effect and a preparation method and application thereof, uses an electron acceptor unit, 6, 6, 12, 12-tetra(4-hexylphenyl)-6, 12-dihydrodithiophene[2, 3-d: 2', 3'-d']-s-indaceno[1, 2-b: 5, 6-b']dithiophene-2, 8-ditrimethyl tin and (4, 4-bis(6-bromohexyl)-4H-cyclopenta[2, 1-b: 3, 4-b']dithiophene-2, 6-diyl) bis(trimethylstannane) as an electron donor unit, carries out a polymerization reaction to obtain an intermediate polymer with an aggregation-induced emission effect; and then a phosphate group is introduced into a side chain of the intermediate polymer to obtain the near-infrared two-region fluorescent conjugated polymer with the anti-aggregation quenching effect. The water-soluble nanoparticles after self-assembly of F-127 can be used as a contrast agent.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanomedicine imaging, and particularly relates to a near-infrared two-region fluorescent conjugated polymer with anti-aggregation quenching effect and a preparation method and application thereof. BACKGROUND

[0002] Cancer is one of the leading causes of death worldwide, with millions of people losing their lives each year due to cancer and its related complications. With the continuous advancement of medical technology, significant progress has been made in the field of cancer treatment. Among them, near-infrared two-region fluorescence imaging (wavelength range: 900-1700 nm) guided two-region photothermal therapy as a new treatment method has attracted much attention due to its high precision and low invasiveness. Photothermal therapy is a non-invasive treatment technology based on photothermal effect, which converts light energy into heat energy through an external light source, thereby precisely destroying target tissues or cells while minimizing damage to surrounding healthy tissues. Near-infrared two-region fluorescence imaging technology has deeper tissue penetration and higher signal-to-noise ratio. This technology has shown great application potential in molecular diagnosis and treatment. Due to the low photon scattering and attenuation characteristics of near-infrared two-region fluorescence imaging technology, it can achieve deeper imaging in biological tissues and provide high temporal resolution and high sensitivity real-time monitoring. This non-invasive imaging technology not only enables more precise tracking of tumor dynamics, but also provides strong support for timely adjustment of treatment plans, thereby significantly promoting the development of precision medicine.

[0003] Therefore, the development of an excellent near-infrared two-region imaging contrast agent that simultaneously realizes near-infrared two-region fluorescence imaging and two-region photothermal therapy has attracted widespread attention. Conjugated polymer luminescent materials have the advantages of durability, strong resistance to photobleaching, and easy-to-control structure, and are widely used in imaging and photodiagnosis and treatment. However, the existing conjugated polymers have the problem of aggregation-induced quenching during the preparation of water-soluble nanoparticles, which leads to a significant reduction in fluorescence intensity in aqueous solution. SUMMARY

[0004] Objective: To solve the problem of fluorescence quenching of conjugated polymer water-soluble nanoparticles, the present application provides a near-infrared two-region fluorescent conjugated polymer with anti-aggregation quenching effect, as well as a near-infrared two-region imaging contrast agent prepared by using the conjugated polymer and a preparation method and use thereof. The conjugated polymer has aggregation-induced emission characteristics that can reduce aggregation quenching, and the polymer side chain is modified with a phosphate group that can enhance the interaction with the embedding agent F-127, thereby solving the fluorescence quenching of the conjugated polymer by water molecules.

[0005] The technical scheme adopted by the present application is as follows:

[0006] In one aspect, the application provides a near-infrared two-region fluorescent conjugated polymer with anti-aggregation quenching effect, and the chemical structure of the polymer is as follows:

[0007]

[0008] wherein m and n represent the number of repeating units, m is 3-10, and n is 3-10.

[0009] In some embodiments, m and n are preferably 5-8.

[0010] In another aspect, the application also provides a preparation method of the near-infrared two-region fluorescent conjugated polymer, comprising:

[0011] S1: using 4,8-Bis(3-(2-ethylhexyl)-2-thiophenyl)-2-benzo-[1,2-c;4,5-c']-Bis[1,2,5]-thiadiazole as an electron acceptor unit, (E)-1,2-bis(5-methylthiophene-2-stannyl) ethylene and (4,4-bis(6-bromohexyl)-4H-cyclopenta[2,1-b:3,4-b']dithiophene-2,6-diyl) bis(trimethylstannane) as an electron donor unit, and performing a polymerization reaction to obtain an intermediate polymer with an aggregation-induced emission effect; wherein the chemical structure of the intermediate polymer is as follows:

[0012]

[0013] S2: reacting the intermediate polymer with triethyl phosphite to obtain the near-infrared two-region fluorescent conjugated polymer.

[0014] In some embodiments, in step S1, the amount-of-substance ratio of the monomers 4,8-Bis(3-(2-ethylhexyl)-2-thiophenyl)-2-benzo-[1,2-c;4,5-c']-Bis[1,2,5]-thiadiazole, 6,6,12,12-tetra(4-hexylphenyl)-6,12-dihydrodithiopheno[2,3-d:2',3'-d']-s-indaceno[1,2-b:5,6-b']dithiophene-2,8-ditrimethyltin, and (4,4-bis(6-bromohexyl)-4H-cyclopenta[2,1-b:3,4-b']dithiophene-2,6-diyl) bis(trimethylstannane) is 2:1:1.

[0015] In some embodiments, in step S1, anhydrous toluene is used as the reaction solvent.

[0016] In some embodiments, in step S1, the reaction conditions are 110°C in an oxygen-free environment for 2-6 hours.

[0017] In some embodiments, in step S1, (dibenzylideneacetone) palladium and triphenylphosphine are added as catalysts during the reaction.​

[0018] In some embodiments, in step S2, the reaction condition is 140℃ for 16 hours.

[0019] The electron acceptor unit of the intermediate polymer is 4,8-Bis(3-(2-ethylhexyl)-2- thiophenyl)-2-benzo-[1,2-c;4,5-c']-Bis[1,2,5]-thiadiazole, and the electron donor unit of the intermediate polymer is 6,6,12,12-tetra(4-hexylphenyl)-6,12-dihydrodithiopheno[2,3-d:2',3'-d']-s-indaceno[1,2-b:5,6-b']dithiophene-2,8-ditrimethyl tin and (4,4-bis(6-bromohexyl)-4H-cyclopenta[2,1-b:3,4-b']dithiophene-2,6-diyl)bis(trimethylstannane).

[0020] In actual preparation, the values of m and n can be adjusted by controlling the reaction time of step S1, and the ratio of m to n can be adjusted by the ratio of the two electron donor units provided in the preparation process.

[0021] In another aspect, the present application also provides the use of the near-infrared two-region fluorescent conjugated polymer in the preparation of a contrast agent.

[0022] In another aspect, the present application also provides a contrast agent comprising the near-infrared two-region fluorescent conjugated polymer.

[0023] The preparation method of the contrast agent comprises the following steps: mixing a tetrahydrofuran solution of the near-infrared two-region fluorescent conjugated polymer with an aqueous solution of the amphiphilic triblock polymer F-127 to obtain a mixed solvent liquid; and then removing excess tetrahydrofuran by dialysis to obtain the contrast agent.

[0024] In some embodiments, the mixing is carried out under room temperature ultrasonic conditions.

[0025] In another aspect, the present application also provides the use of the contrast agent in the preparation of a tumor disease diagnosis and treatment drug.

[0026] In some embodiments, the present application also provides a method for preparing a contrast agent using the near-infrared two-region fluorescent conjugated polymer with anti-aggregation quenching effect, comprising the following steps:

[0027] S01 dissolving the near-infrared two-region fluorescent conjugated polymer in tetrahydrofuran to obtain a tetrahydrofuran solution of the near-infrared two-region fluorescent conjugated polymer;

[0028] S02 dissolving the amphiphilic triblock polymer F-127 in water to obtain an aqueous solution of the amphiphilic triblock polymer F-127;

[0029] S03 mixing the tetrahydrofuran solution of the near-infrared two-region fluorescent conjugated polymer with the aqueous solution of the amphiphilic triblock polymer F-127 to obtain a mixed solvent liquid;

[0030] S04 removing the excess tetrahydrofuran by dialysis.

[0031] Specifically, the step S03 is performed under room temperature ultrasonic conditions.

[0032] In the process of preparing the tetrahydrofuran solution of the near-infrared two-region fluorescent conjugated polymer, the mass-volume ratio of the near-infrared two-region fluorescent conjugated polymer to tetrahydrofuran is 0.1-1.0 mg / ml.

[0033] In the process of preparing the aqueous solution of the amphiphilic triblock polymer F-127, the mass-volume ratio of the amphiphilic triblock polymer F-127 to water is 1-20 mg / ml.

[0034] In the process of preparing the contrast agent, the mass ratio of the near-infrared two-region fluorescent conjugated polymer to the amphiphilic triblock polymer F-127 is 1:5-1:100.

[0035] The beneficial effects of the present application include: the present application designs, synthesizes and prepares a near-infrared two-region fluorescent conjugated polymer with anti-aggregation quenching effect, which is obtained by polymerization reaction based on using 4,8-Bis(3-(2-ethylhexyl)-2-thienyl)-2-benzo-[1,2-c;4,5-c']-Bis[1,2,5]-thiadiazole as an electron acceptor unit, 6,6,12,12-tetra(4-hexylphenyl)-6,12-dihydrodithieno[2,3-d:2',3'-d']-s-indaceno[1,2-b:5,6-b']dithiophene-2,8-ditrimethyl tin and (4,4-bis(6-bromohexyl)-4H-cyclopenta[2,1-b:3,4-b']dithiophene-2,6-diyl)bis(trimethylstannane) as an electron donor unit to obtain an intermediate polymer with aggregation-induced emission effect; then introducing a phosphate group into the side chain of the intermediate polymer to obtain a near-infrared two-region fluorescent conjugated polymer with anti-aggregation quenching effect. The synthesis route and preparation method are very simple. The conjugated polymer first has the aggregation-induced emission characteristic, which can reduce aggregation quenching, and the side chain of the polymer is modified with a phosphate group, which can enhance the interaction with the embedding agent F-127, solving the fluorescence quenching of the conjugated polymer by water molecules. The synthesized conjugated polymer and the amphiphilic block polymer F-127 self-assemble to obtain water-soluble nanoparticles with excellent near-infrared two-region luminescence effect, which can be used as a contrast agent to realize near-infrared two-region fluorescence imaging, and can also be used for near-infrared two-region excitation photothermal therapy to realize the diagnosis and treatment integration effect on tumors. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 Transmission electron microscope photo of the near-infrared two-region fluorescent nanometer contrast agent with anti-aggregation quenching effect obtained in Example 1 of the present application;

[0037] Figure 2 Absorption spectrum of the near-infrared two-region fluorescent nanometer contrast agent with anti-aggregation quenching effect obtained in Example 1 of the present application;

[0038] Figure 3 Fluorescence spectrum of the near-infrared two-region fluorescent nanometer contrast agent with anti-aggregation quenching effect obtained in Example 1 of the present application;

[0039] Figure 4 Absorption spectrum of the near-infrared two-region fluorescent nanometer contrast agent with anti-aggregation quenching effect obtained in Example 2 of the present application;

[0040] Figure 5 Fluorescence spectrum of the near-infrared two-region fluorescent nanometer contrast agent with anti-aggregation quenching effect obtained in Example 2 of the present application;

[0041] Figure 6 Absorption spectrum of the near-infrared two-region fluorescent nanometer contrast agent with anti-aggregation quenching effect obtained in Example 2 of the present application;

[0042] Figure 7 Fluorescence spectrum of the near-infrared two-region fluorescent nanometer contrast agent with anti-aggregation quenching effect obtained in Example 1 of the present application;

[0043] Figure 8 Near-infrared two-region imaging photo of the near-infrared two-region fluorescent nanometer contrast agent with anti-aggregation quenching effect obtained in Example 1 of the present application on mice;

[0044] Figure 9 In-vivo photothermal therapy thermal imaging photo of the near-infrared two-region fluorescent nanometer contrast agent with anti-aggregation quenching effect obtained in Example 2 of the present application;

[0045] Figure 10 Nuclear magnetic resonance photo of the conjugated polymer prepared in the present application. DETAILED DESCRIPTION

[0046] In order to further illustrate the present application, a series of examples are given below, which are purely illustrative and are only used to specifically describe the present application, and should not be understood as limiting the present application.

[0047] Synthetic method of the conjugated polymer:

[0048] The monomer 4,8-Bis(3-(2-ethylhexyl)-2-thienyl)-2-benzo-[1,2-c;4,5-c']-bis[1,2,5]-thiadiazole is reacted with 6,6,12,12-tetra(4-hexylphenyl)-6,12-dihydrodithiopheno[2,3-d:2',3'-d']-s-indaceno[1,2-b:5,6-b']dithiophene-2,8-ditrimethyl tin and (4,4-bis(6-bromohexyl)-4H-cyclopenta[2,1-b:3,4-b']dithiophene-2,6-diyl)bis(trimethylstannane) in a molar ratio of 2:1:1, dissolved in 3-5 mL of anhydrous toluene, and reacted at 110°C in an oxygen-free environment for 2-6 hours to obtain an intermediate polymer. Among them, (dibenzylideneacetone) palladium (3.0 micromoles, 2.75 milligrams) and triphenylphosphine (12 micromoles, 3.15 milligrams) are used as catalysts for the reaction. After the reaction is completed, the reaction solution is cooled, and then poured into a large amount of methanol to precipitate. The precipitate is collected and dried in a vacuum drying box to obtain an intermediate polymer. The intermediate polymer is then reacted with excess triethyl phosphite at 140°C for 16 hours. After the reaction is completed, the reaction solution is cooled, and then poured into a large amount of methanol to precipitate and dry to obtain a conjugated polymer. The nuclear magnetic resonance chart is shown in Figure 10 .

[0049] As Figure 2 and Figure 3 shown, the absorption and emission spectrum of the conjugated polymer is tested. It can be seen that the ultraviolet-near infrared absorption peak is at a wavelength of 731 nanometers, and the emission peak is at a wavelength of 1111 nanometers. There is also an emission shoulder peak at about 1279 nanometers, which is obviously a two-window emission material. It can be seen from Figure 6 that as the proportion of water in the solution gradually rises, the fluorescence intensity of the conjugated polymer also rises, which reflects the excellent AIE performance of the conjugated polymer.

[0050] The synthesis route is as follows:

[0051] Example

[0052] 0.5 milligrams of the conjugated polymer is dissolved in 1 milliliter of tetrahydrofuran, and then 30 milligrams of the amphiphilic triblock polymer F-127 is dissolved in 10 milliliters of water. Under ultrasonic conditions at room temperature, the tetrahydrofuran solution of the dissolved conjugated intermediate polymer is directly added to the F-127 aqueous solution to obtain a yellow-green mixed solvent liquid. The excess tetrahydrofuran is removed by dialysis to obtain the contrast agent.

[0053] A contrast agent aqueous solution with a conjugated polymer concentration of 0.02 milligrams / milliliter is prepared, and the microstate of the contrast agent is observed by transmission electron microscopy, as shown in Figure 1As shown, the contrast agent nanoparticles have a diameter of about 100 nanometers and are all spherical.

[0054] Prepare an aqueous solution of contrast agent with a conjugated polymer concentration of 0.1 mg / mL, and test its absorption spectrum, such as... Figure 2 As shown, the absorption peak is in the 731 nm band.

[0055] Prepare an aqueous contrast agent solution with a conjugated polymer concentration of 0.1 mg / mL, and test its fluorescence spectrum as shown below. Figure 3 As shown, the emission peak is at 1111 nm and the emission shoulder peak is at 1279 nm, proving that it is a two-window emission material.

[0056] A contrast agent aqueous solution with a conjugated polymer concentration of 2 mg / mL was prepared. MCF-7 nude mice with axillary tumors were selected, and 120 mL of the contrast agent was injected via the tail vein. The signal in the mouse abdomen was then observed using a near-infrared spectroscopy system (NIR 2D). Figure 7 As shown, the distribution of blood vessels can be clearly observed with high resolution.

[0057] like Figure 8 As shown, a contrast agent aqueous solution with a conjugated polymer concentration of 2 mg / mL was prepared. MCF-7 nude mice with axillary tumors were selected, and 120 mL of the contrast agent was injected via the tail vein. After 24 hours, the tumors were irradiated with a 1064 nm LED lamp. The temperature of the mouse tumors was monitored using a thermal imager, and thermal images of the mice during photothermal therapy were obtained as shown. Figure 8 As shown, this contrast agent has excellent photothermal therapy effects. Example

[0058] 0.5 mg of the conjugated polymer was dissolved in 2 mL of tetrahydrofuran, and 20 mg of the amphiphilic triblock polymer F-127 was dissolved in 10 mL of water. Under room temperature sonication, the tetrahydrofuran solution of the dissolved conjugated polymer was directly added to the F-127 aqueous solution, resulting in a dark green mixed solvent liquid. Excess tetrahydrofuran was then removed by dialysis to obtain the contrast agent.

[0059] Prepare an aqueous solution of contrast agent with a conjugated polymer concentration of 0.1 mg / mL, and test its absorption spectrum, such as... Figure 4 As shown, the absorption peak is in the 737 nm band.

[0060] Prepare an aqueous contrast agent solution with a conjugated polymer concentration of 0.1 mg / mL and test its fluorescence spectrum, such as... Figure 5 As shown, the emission peak is in the 1096 nm band and the emission shoulder peak is in the 1276 nm band, proving that it is a two-window emission material.

[0061] The process of preparing an aqueous solution of the conjugated polymer tetrahydrofuran and testing its AIE effect is shown in the figure below.Figure 6 As shown in the figure, under the irradiation of 808 nm laser, the fluorescence intensity of the conjugated polymer solution gradually increases with the increase of the water content in the tetrahydrofuran / water mixture, indicating that the conjugated polymer has excellent AIE effect.

[0062] The conjugated polymer concentration of the contrast agent aqueous solution is 2 mg / ml, MCF-7 axillary tumor nude mice are selected, and after 120 ml of the contrast agent is injected into the tail vein, the obtained in vivo imaging figure of the near-infrared two-zone contrast agent is shown in the figure Figure 9 As shown in the figure, it can be seen that after 24-36 hours of tail vein injection, the contrast agent is obviously enriched in the tumor site, and the near-infrared two-zone fluorescence intensity is obviously higher than that of the surrounding tissue, so the contrast agent has good near-infrared two-zone fluorescence imaging effect.

[0063] The above only describes the preferred embodiments of the present application, and it should be pointed out that for ordinary skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A near-infrared two-region fluorescent conjugated polymer with an anti-aggregation quenching effect, characterized in that, The chemical structure of the polymer is as follows: ； Wherein, m, n represent the number of repeating units, m is 3~10, n is 3~10.

2. The near-infrared two-region fluorescent conjugated polymer according to claim 1, characterized in that, Both m and n are 5~8.

3. The method of producing the near-infrared two-region fluorescent conjugated polymer according to claim 1, characterized by, Comprise: S1: using 4, 8-Bis(3-(2-ethylhexyl)-2-thiophenyl)-2-benzo-[1, 2-c; 4, 5-c']-Bis[1, 2, 5]-thiadiazole as an electron acceptor unit, 6, 6, 12, 12-tetrakis(4-hexylphenyl)-6, 12-dihydrodithiophene[2, 3-d: 2', 3'-d']-s-indaceno[1, 2-b: 5, 6-b']dithiophene-2, 8-ditrimethyl tin and (4, 4-bis(6-bromohexyl)-4H-cyclopenta[2, 1-b: 3, 4-b']dithiophene-2, 6-diyl) bis(trimethylstannane) as an electron donor unit, polymerization is carried out to obtain an intermediate polymer with aggregation induced emission effect; wherein the chemical structure of the intermediate polymer is as follows: ； S2: reacting the intermediate polymer with triethyl phosphite to obtain the near-infrared two-region fluorescent conjugated polymer.

4. The method of producing the near-infrared two-region fluorescent conjugated polymer according to claim 3, characterized by, In step S1, the mass ratio of the monomers 4, 8-Bis(3-(2-ethylhexyl)-2-thiophenyl)-2-benzo-[1, 2-c; 4, 5-c']-Bis[1, 2, 5]-thiadiazole, 6, 6, 12, 12-tetrakis(4-hexylphenyl)-6, 12-dihydrodithiophene[2, 3-d: 2', 3'-d']-s-indaceno[1, 2-b: 5, 6-b']dithiophene-2, 8-ditrimethyl tin and (4, 4-bis(6-bromohexyl)-4H-cyclopenta[2, 1-b: 3, 4-b']dithiophene-2, 6-diyl) bis(trimethylstannane) is 2:1:

1.

5. The preparation method of the near-infrared two-region fluorescent conjugated polymer of claim 3, wherein, And / or, in step S1, anhydrous toluene is used as the reaction solvent; And / or, in step S1, the reaction condition is 110℃ under oxygen-free environment for 2-6 hours; And / or, in step S1, (dibenzylideneacetone) palladium and triphenylphosphine are added as catalysts during the reaction.

6. The use of the near-infrared two-region fluorescent conjugated polymer of claim 1 or 2 in the preparation of a contrast agent.

7. A contrast agent comprising the near-infrared two-region fluorescent conjugated polymer of claim 1 or 2.

8. The method of claim 7, wherein the contrast agent is prepared by the process comprising the steps of: Comprise: The tetrahydrofuran solution of the near-infrared two-region fluorescent conjugated polymer is mixed with the aqueous solution of the amphiphilic triblock polymer F-127 to obtain a mixed solvent liquid; then excess tetrahydrofuran is removed by dialysis to obtain the contrast agent.

9. The preparation method of the contrast agent of claim 8, wherein, The mixing is carried out under room temperature ultrasonic condition; And / or, in the contrast agent, the mass ratio of the near-infrared two-region fluorescent conjugated polymer to the amphiphilic triblock polymer F-127 is 1:5~1:

100.

10. The use of the contrast agent of any one of claims 7 to 9 in the preparation of a drug for the diagnosis and treatment of tumor diseases.

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