Near-infrared two-region fluorescent conjugated polymer with anti-aggregation quenching effect as well as preparation method and application of near-infrared two-region fluorescent conjugated polymer

Abstract

The invention discloses a near-infrared two-region fluorescent conjugated polymer with anti-aggregation quenching effect and a preparation method and application thereof.The near-infrared two-region fluorescent conjugated polymer is prepared from an electron acceptor unit, 6, 6, 12, 12-tetrakis (4-hexylphenyl)-6, 12-dihydrodithiophene [2, 3-d: 2 ', 3'-d ']-s-indeno [1, 2-b: 5, 6-b'] dithiophene-2, 8-di-trimethyltin and (4, 4-bis (6-bromohexyl)-4H-cyclopenta [2, 1-b: 3, 4-b '] dithiophene-2, 8-di-trimethyltin), 2, 6-diyl) bis (trimethylstannane) is used as an electron donor unit, and a polymerization reaction is performed to obtain an intermediate polymer with an aggregation-induced emission effect; and introducing a phosphate group into the 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 nano-particles which are self-assembled with F-127 can be used as a contrast agent.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomedicine imaging, and relates to a polymer, in particular to a near-infrared II region fluorescent conjugated polymer with an anti-aggregation quenching effect, and a preparation method and application thereof. Background Art

[0002] Cancer is one of the main factors leading to death globally, and millions of people lose their lives every year due to cancer and its related complications. With the continuous progress of medical technology, significant progress has been made in the field of cancer treatment. Among them, near-infrared II region fluorescence imaging (wavelength range: 900 - 1700 nm)-guided photothermal therapy in the second region, as an emerging treatment method, has attracted much attention due to its high precision and low invasiveness. Photothermal therapy is a non-invasive treatment technology based on the 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 II region fluorescence imaging technology has deeper tissue penetration ability and higher signal-to-noise ratio. This technology shows great application potential in molecular diagnosis and treatment. Due to the characteristics of low photon scattering and attenuation of near-infrared II region fluorescence imaging technology, it can achieve deeper imaging in biological tissues and provide real-time monitoring with high time resolution and high sensitivity. This non-invasive imaging technology can not only more accurately track the dynamic changes of tumors but also provide strong support for the timely adjustment of treatment plans, thus significantly promoting the development of precision medicine.

[0003] Therefore, the development of an excellent near-infrared II region imaging contrast agent that can simultaneously achieve near-infrared II region fluorescence imaging and photothermal therapy in the second region has attracted extensive attention. Conjugated polymer luminescent materials have advantages such as durability, strong anti-photobleaching ability, and easy structural regulation, and are widely used in imaging and photodiagnosis and treatment. However, the existing conjugated polymers currently have the problem of aggregation-induced quenching during the preparation of water-soluble nanoparticles, resulting in a significant reduction in their fluorescence intensity in the aqueous phase. Summary of the Invention

[0004] Objective: To solve the problem of fluorescence quenching of water-soluble nanoparticles of conjugated polymers, the present invention provides a near-infrared II region fluorescent conjugated polymer with an anti-aggregation quenching effect, and a method for preparing a near-infrared II region imaging contrast agent using this conjugated polymer, as well as its preparation method and uses. This conjugated polymer first has the characteristic of aggregation-induced emission, which can reduce aggregation quenching. At the same time, the polymer side chain is modified with phosphate groups, which can enhance the interaction with the embedding agent F-127 and solve the fluorescence quenching of water molecules to the conjugated polymer.

[0005] The technical solution adopted by the present invention is as follows: On the one hand, a near-infrared second-region fluorescent conjugated polymer with an anti-aggregation quenching effect is provided. The general chemical structure formula of the polymer is:

[0006] wherein m and n represent the number of repeating units, m is 3 to 10, and n is 3 to 10.

[0007] In some embodiments, m and n are preferably 5 to 8.

[0008] On the other hand, the present invention also provides a preparation method of the near-infrared second-region fluorescent conjugated polymer, including: S1: 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, (E)-1,2-bis(5-methylthiophen-2-yl)ethylene and (4,4-bis(6-bromohexyl)-4H-cyclopenta[2,1-b:3,4-b']dithiophene-2,6-diyl)bis(trimethylstannane) as electron donor units, and performing a polymerization reaction to obtain an intermediate polymer with an aggregation-induced emission effect; wherein the chemical structural formula of the intermediate polymer is: ; S2: Reacting the intermediate polymer with triethyl phosphite to obtain the product.

[0009] In some embodiments, in step S1, the molar ratio of the monomer 4,8-Bis(3-(2-ethylhexyl)-2-thienyl)-2-benzo-[1,2-c;4,5-c']-Bis[1,2,5]-thiadiazole to 6,6,12,12-tetrakis(4-hexylphenyl)-6,12-dihydrodithieno[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.

[0010] In some embodiments, in step S1, the reaction solvent is anhydrous toluene.

[0011] In some embodiments, in step S1, the reaction conditions are to react for 2 to 6 hours in an oxygen-free environment at 110 °C.

[0012] In some embodiments, in step S1, during the reaction process, (dibenzylideneacetone)palladium(II) and triphenylphosphine are added as catalysts.

[0013] In some embodiments, in step S2, the reaction conditions are to react at 140 °C for 16 hours.

[0014] The electron acceptor unit of the above intermediate polymer is: 4,8 - Bis(3-(2 - ethylhexyl)-2 - thienyl)-2 - benzo - [1,2 - c;4,5 - c']-Bis[1,2,5]-thiadiazole; the electron donor units of the intermediate polymer are: 6,6,12,12 - tetra(4 - hexylphenyl)-6,12 - dihydrodithieno[2,3 - d:2',3' - d']-s - indeno[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).

[0015] In the actual preparation process, the sizes 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.

[0016] On the other hand, the present invention also provides the application of the above - mentioned near - infrared second - region fluorescent conjugated polymer in the preparation of a contrast agent.

[0017] On the other hand, the present invention also provides a contrast agent comprising the above - mentioned near - infrared second - region fluorescent conjugated polymer.

[0018] The preparation method of the contrast agent includes: mixing a tetrahydrofuran solution of the near - infrared second - region fluorescent conjugated polymer with an aqueous solution of the amphiphilic triblock polymer F - 127 to obtain a mixed solvent liquid; then removing the excess tetrahydrofuran by dialysis to obtain the contrast agent.

[0019] In some embodiments, the mixing is carried out under ultrasonic conditions at room temperature.

[0020] On the other hand, the present invention also provides the application of the above - mentioned contrast agent in the preparation of drugs for the diagnosis and treatment of tumor diseases.

[0021] In some embodiments, a method for preparing a contrast agent using the above - mentioned near - infrared second - region fluorescent conjugated polymer with an anti - aggregation quenching effect is also provided, including the following steps: S01 Dissolve the near - infrared second - region fluorescent conjugated polymer in tetrahydrofuran to obtain a tetrahydrofuran solution of the near - infrared second - region fluorescent conjugated polymer; S02 Dissolve the amphiphilic triblock polymer F - 127 in water to obtain an aqueous solution of the amphiphilic triblock polymer F - 127; S03 Mix the tetrahydrofuran solution of the near - infrared second - region fluorescent conjugated polymer with the aqueous solution of the amphiphilic triblock polymer F - 127 to obtain a mixed solvent liquid; S04 uses dialysis to remove excess tetrahydrofuran.

[0022] Specifically, step S03 is performed under ultrasonic conditions at room temperature.

[0023] In the process of preparing the tetrahydrofuran solution of the near-infrared second-zone fluorescent conjugated polymer, the mass volume ratio of the near-infrared second-zone fluorescent conjugated polymer to tetrahydrofuran is 0.1-1.0 mg / ml.

[0024] 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.

[0025] During the preparation of the contrast agent, the mass ratio of the near-infrared second-zone fluorescent conjugated polymer to the amphiphilic triblock polymer F-127 is 1:5~1:100.

[0026] The beneficial effects of the present invention include: the present invention designs, synthesizes and prepares a near-infrared second-zone fluorescent conjugated polymer with anti-aggregation quenching effect, which is based on the use of 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-tetrakis(4-hexylphenyl)-6,12-dihydrodithiophene[2,3-d:2',3' -d']-s-indeno[1,2-b:5,6-b']dithiophene-2,8-ditrimethyltin and (4,4-bis(6-bromohexyl)-4H-cyclopentadien[2,1-b:3,4-b']dithiophene-2,6-diyl)bis(trimethyltin) are used as electron donor units to carry out polymerization reaction to obtain an intermediate polymer with aggregation-induced emission effect; then phosphate groups are introduced into the side chains of the intermediate polymer to obtain a near-infrared second-zone fluorescent conjugated polymer with anti-aggregation quenching effect. Both the synthetic route and the preparation method are very simple. First, the conjugated polymer has the characteristics of aggregation-induced emission, which can reduce aggregation quenching. At the same time, the polymer side chain is modified with phosphate groups, which can enhance the interaction with the embedding agent F-127 and solve the fluorescence quenching of the conjugated polymer by water molecules. The water-soluble nanoparticles with excellent near-infrared zone II luminescence effect obtained by self-assembly of the synthesized conjugated polymer and the amphiphilic block polymer F-127 can be used as contrast agents to realize near-infrared zone II fluorescence imaging, and can also be used for near-infrared zone II excitation photothermal therapy to achieve an integrated diagnosis and treatment effect for tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a transmission electron microscope photograph of the near-infrared second-zone fluorescent nanocontrast agent with anti-aggregation quenching effect obtained in Example 1 of the present invention; Figure 2 This is the absorption spectrum of the near-infrared second-region fluorescent nano-contrast agent with anti-aggregation quenching effect obtained in Example 1 of the present invention; Figure 3 The fluorescence spectrum of the near-infrared second-region fluorescent nano-contrast agent with anti-aggregation quenching effect obtained in Example 1 of the present invention; Figure 4 This is the absorption spectrum of the near-infrared second-region fluorescent nano-contrast agent with anti-aggregation quenching effect obtained in Example 2 of the present invention; Figure 5 The fluorescence spectrum of the near-infrared second-region fluorescent nano-contrast agent with anti-aggregation quenching effect obtained in Example 2 of the present invention; Figure 6 This is the absorption spectrum of the near-infrared second-region fluorescent nano-contrast agent with anti-aggregation quenching effect obtained in Example 2 of the present invention; Figure 7 The fluorescence spectrum of the near-infrared second-region fluorescent nano-contrast agent with anti-aggregation quenching effect obtained in Example 1 of the present invention; Figure 8 This is a near-infrared zone II imaging image of a mouse by the near-infrared zone II fluorescent nano contrast agent with anti-aggregation quenching effect obtained in Example 1 of the present invention; Figure 9 This is a thermal imaging photograph of the in vivo photothermal therapy of the near-infrared second-zone fluorescent nano-contrast agent with anti-aggregation quenching effect obtained in Example 2 of the present invention; Figure 10 This is the NMR image of the conjugated polymer prepared in the embodiment of the present invention. DETAILED DESCRIPTION

[0028] In order to further illustrate the present invention, a series of examples are given below. These examples are purely illustrative and are only used to specifically describe the present invention, and should not be understood as limiting the present invention.

[0029] Synthesis of conjugated polymers: The monomer 4,8-Bis(3-(2-ethylhexyl)-2-thienyl)-2-benzo-[1,2-c;4,5-c']-Bis[1,2,5]-thiadiazole, 6,6,12,12-tetrakis(4-hexylphenyl)-6,12-dihydrodithieno[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) were dissolved in 3 - 5 mL of anhydrous toluene in a molar ratio of 2:1:1 and reacted in an oxygen-free environment at 110 °C for 2 - 6 hours to obtain an intermediate polymer. Among them, bis(dibenzylideneacetone)palladium (0) (3.0 μmol, 2.75 mg) and triphenylphosphine (12 μmol, 3.15 mg) were used as the catalysts for this reaction. After the reaction was completed, the reaction solution was allowed to cool, and then poured into a large amount of methanol for precipitation. The precipitate was collected and dried in a vacuum drying oven to obtain the intermediate polymer. Then, the intermediate polymer was reacted with an excess of triethyl phosphite at 140 °C for 16 hours. After the reaction was completed, the reaction solution was allowed to cool, and then poured into a large amount of methanol for precipitation and drying to obtain the conjugated polymer. The NMR spectrum is as shown in Figure 10 shown.

[0030] As Figure 2 shown in Figure 3 and shown in Figure 6 , by testing its absorption and emission spectra, it can be seen that its ultraviolet-near infrared absorption peak is in the 731 nm band, while the emission peak is in the 1111 nm band, and there is also an emission shoulder peak at about 1279 nm, which is obviously a two-window emission material. As can be seen from

[0031] , as the proportion of water in the solution gradually increases, the fluorescence intensity of the conjugated polymer also increases, which reflects that the conjugated polymer has excellent AIE performance.

[0031] The synthesis route is as follows: Example

[0032] 0.5 mg of the conjugated polymer was dissolved in 1 mL of tetrahydrofuran, and 30 mg of the amphiphilic triblock polymer F-127 was dissolved in 10 mL of water. Under ultrasonic conditions at room temperature, the tetrahydrofuran solution of the dissolved conjugated intermediate polymer was directly added to the F-127 aqueous solution to obtain a yellow-green mixed solvent liquid. Then, the excess tetrahydrofuran was removed by dialysis to obtain the contrast agent.

[0033] An aqueous solution of the contrast agent with a conjugated polymer concentration of 0.02 mg / mL was prepared, and the microscopic state of the contrast agent was observed with a transmission electron microscope. As shown in Figure 1 shown, it can be seen that the diameter of the contrast agent nanoparticles is about 100 nm and they are all spherical.

[0034] Prepare an aqueous solution of the contrast agent with a conjugated polymer concentration of 0.1 mg / mL and test its absorption spectrum. As Figure 2 shown, it can be seen that the absorption peak is in the 731 nm band.

[0035] Prepare an aqueous solution of the contrast agent with a conjugated polymer concentration of 0.1 mg / mL and test its fluorescence spectrum as Figure 3 shown. It can be seen that the emission peak is in the 1111 nm band and the emission shoulder peak is in the 1279 nm band, proving that it is a two-window emission material.

[0036] Prepare an aqueous solution of the contrast agent with a conjugated polymer concentration of 2 mg / mL. Select nude mice with MCF-7 axillary tumors. After tail vein injection of 120 mL of the contrast agent, observe the signals in the abdomen of the mice with a second near-infrared imager. As Figure 7 shown, the blood vessel distribution can be clearly observed with high resolution.

[0037] As Figure 8 shown, prepare an aqueous solution of the contrast agent with a conjugated polymer concentration of 2 mg / mL. Select nude mice with MCF-7 axillary tumors. After tail vein injection of 120 mL of the contrast agent, after 24 hours, irradiate the tumor with a 1064 nm LED lamp and monitor the temperature of the mouse tumor with a thermal imager to obtain the thermal imaging photo of the mouse during photothermal therapy as Figure 8 shown. It can be seen that the contrast agent has excellent photothermal therapy effects. Example

[0038] Dissolve 0.5 mg of the conjugated polymer in 2 mL of tetrahydrofuran, and then dissolve 20 mg of the amphiphilic triblock polymer F-127 in 10 mL of water. Under ultrasonic conditions at room temperature, directly add the dissolved tetrahydrofuran solution of the conjugated polymer to the F-127 aqueous solution to obtain a dark green mixed solvent liquid. Then remove the excess tetrahydrofuran by dialysis to obtain the contrast agent.

[0039] Prepare an aqueous solution of the contrast agent with a conjugated polymer concentration of 0.1 mg / mL and test its absorption spectrum. As Figure 4 shown, it can be seen that the absorption peak is in the 737 nm band.

[0040] Prepare an aqueous solution of the contrast agent with a conjugated polymer concentration of 0.1 mg / mL and test its fluorescence spectrum. As Figure 5 shown, it can be seen that 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.

[0041] Prepare an aqueous solution of the conjugated polymer in tetrahydrofuran and test its AIE effect process diagram as Figure 6As shown, under the irradiation of 808 nm laser, with the increase of the water content in the tetrahydrofuran / water mixture, the fluorescence intensity of the conjugated polymer solution gradually increases, indicating that the conjugated polymer has excellent AIE effect.

[0042] Prepare an aqueous solution of the contrast agent with a conjugated polymer concentration of 2 mg / mL. Select nude mice with MCF-7 axillary tumors. After injecting 120 mL of the contrast agent via the tail vein, the in vivo imaging diagram of the near-infrared second-region contrast agent obtained by real-time imaging is as Figure 9 shown. By observing different time points, it can be seen that 24-36 hours after the tail vein injection, the contrast agent is significantly enriched at the tumor site, and the near-infrared second-region fluorescence intensity is significantly higher than that of the surrounding tissues. It can be seen that the contrast agent has good near-infrared second-region fluorescence imaging effect.

[0043] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A near-infrared second-zone fluorescent conjugated polymer with anti-aggregation quenching effect, characterized in that: The general chemical structure formula of the polymer is: ； Wherein, m and n represent the number of repeating units, m is 3~10, and n is 3~10.

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

3. The method for preparing the near-infrared second-region fluorescent conjugated polymer according to claim 1, characterized in that: include: S1: 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, (E)-1,2-bis(5-methylthiophene-2-tinyl)ethylene and (4,4-bis(6-bromohexyl)-4H-cyclopentadien[2,1-b:3,4-b']dithiophene-2,6-diyl)bis(trimethylstannane) as electron donor units, a polymerization reaction is carried out to obtain an intermediate polymer with aggregation-induced emission effect; wherein the chemical structure of the intermediate polymer is: ； S2: The intermediate polymer is reacted with triethyl phosphite to obtain.

4. The method for preparing the near-infrared second-region fluorescent conjugated polymer according to claim 3, characterized in that: In step S1, the molar ratio of monomers 4,8-Bis(3-(2-ethylhexyl)-2-thienyl)-2-benzo-[1,2-c;4,5-c']-Bis[1,2,5]-thiadiazole and 6,6,12,12-tetrakis(4-hexylphenyl)-6,12-dihydrodithiophene[2,3-d:2',3'-d']-s-indeno[1,2-b:5,6-b']dithiophene-2,8-ditrimethyltin and (4,4-bis(6-bromohexyl)-4H-cyclopentadien[2,1-b:3,4-b']dithiophene-2,6-diyl)bis(trimethyltin) is 2:1:

1.

5. The method for preparing the near-infrared second-region fluorescent conjugated polymer according to claim 3, characterized in that: And / or, in step S1, the reaction solvent is anhydrous toluene; And / or, in step S1, the reaction conditions are 110° C. in an oxygen-free environment for 2-6 hours; And / or, in step S1, during the reaction, (dibenzylideneacetone) dipalladium and triphenylphosphine are added as catalysts.

6. Use of the near-infrared second-zone fluorescent conjugated polymer according to claim 1 or 2 in the preparation of contrast agents.

7. A contrast agent comprising the near-infrared second-region fluorescent conjugated polymer according to claim 1 or 2.

8. The method for preparing a contrast agent according to claim 7, characterized in that: include: The tetrahydrofuran solution of the near-infrared second-zone fluorescent conjugated polymer and the aqueous solution of the amphiphilic triblock polymer F-127 are mixed to obtain a mixed solvent liquid; then, the excess tetrahydrofuran is removed by dialysis to obtain a contrast agent.

9. The method for preparing a contrast agent according to claim 8, characterized in that: The mixing is carried out under ultrasonic conditions at room temperature; And / or, in the contrast agent, the mass ratio of the near-infrared second-zone fluorescent conjugated polymer to the amphiphilic triblock polymer F-127 is 1:5 to 1:

100.

10. Use of the contrast agent according to any one of claims 7 to 9 in the preparation of drugs for diagnosing and treating tumor diseases.

Images