D-A type conjugated polymer based on click chemistry as well as preparation method and application of D-A type conjugated polymer

Abstract

The invention provides a D-A type conjugated polymer based on click chemistry as well as a preparation method and application of the D-A type conjugated polymer. A structure which has strong electron donating ability and strong electron deficiency ability and contains a modification site is selected, a click site is introduced into the modification site of the structure, and then a hydrophilic group and a polymer main chain are covalently bound by utilizing click chemistry, so that the polymer has excellent solubility and stability in an aqueous solution, and the hydrophilic group is not prone to being oxidized. The absorption peak extends to an NIR-II region window, so that long-acting and stable NIR-II region PA imaging is realized. According to the invention, the polymer is self-assembled to form water-soluble nanoparticles, in-vitro photo-thermal and photo-acoustic experimental tests show that the self-assembled water-soluble nanoparticles have excellent photo-thermal performance and photo-acoustic effect, can be stored in an aqueous solution for a long time without aggregation and cracking, and have good biocompatibility; the method can be applied to in-vivo photoacoustic imaging of mice.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and particularly relates to a D-A type conjugated polymer based on click chemistry, a preparation method thereof, and an application thereof. Background Art

[0002] Photoacoustic Imaging (PAI) is a composite imaging technology that combines optical excitation and ultrasonic detection. This technology can effectively promote in-vivo imaging. It has advantages such as excellent tissue penetration ability, high image contrast, and precise spatial resolution. In particular, when using an excitation light source in the second near-infrared region (NIR-II, 1000 - 1700 nm) for PAI, due to the low absorption and scattering of light in this wavelength band, the signal-to-noise ratio can be significantly improved, and deeper imaging penetration can be achieved, thus greatly improving the imaging quality. Based on these advantages, the NIR-II window photoacoustic imaging technology is expected to be clinically applied in the fields of tumor detection and cardiovascular disease imaging, showing broad application prospects.

[0003] Semiconductor polymers have shown great application potential in the biomedical field due to their unique optical properties and easy modification. However, most of these polymers are insoluble in water due to their backbone structures and often require post-modification. For example, semiconductor polymers are encapsulated into amphiphilic polymers through microemulsion or nanoprecipitation to prepare water-dispersible nanoparticles, so as to achieve their solubility, long-term stability, and biological applications in aqueous solutions. This method has made great progress in recent decades. However, this preparation method has its limitations. It has been reported in the literature that amphiphilic copolymers leak from the nanoparticles, thus aggregating in the blood circulation, ultimately resulting in poor imaging effects and biological toxicity. In addition, the nanoparticles interact with proteins or other substances in the body, accelerating the disintegration of the nanoparticles. This change may cause changes in their optical properties and poor biodistribution.

[0004] In order to overcome these potential defects and improve their in-vivo performance, there are many other methods that can successfully prepare nanoparticles, such as solution self-assembly and microfluidic methods. However, designing optical imaging nanoparticles with good stability, consistent size, and small batch-to-batch differences remains a major challenge. In order to overcome the problem of poor solubility of semiconductor polymers in solvents such as aqueous solutions and thus break through their limitations in in-vivo bioimaging applications, a new technical solution is urgently needed to solve the problems existing in the prior art. Summary of the Invention

[0005] Based on this, the present invention provides a click chemistry-based D-A type conjugated polymer and its preparation method and application. The present invention selects a structure with strong electron-donating ability, strong electron-withdrawing ability and containing a modification site. By introducing click sites at the modification site of this structure, and then using click chemistry to covalently bind hydrophilic groups to the polymer backbone, the polymer has excellent solubility and stability in aqueous solution, extends its absorption peak to the NIR-II region window, and realizes long-term stable NIR-II region PA imaging. The present invention also forms water-soluble nanoparticles by self-assembling the polymer. Through in vitro photothermal and photoacoustic experiments, the self-assembled water-soluble nanoparticles have excellent photothermal performance and photoacoustic effects, can be stored in aqueous solution for a long time without aggregation and cleavage, and have good biocompatibility, and can be applied to photoacoustic imaging in mice.

[0006] An object of the present invention is to provide a click chemistry-based D-A type conjugated polymer, and the click chemistry-based D-A type conjugated polymer has the following structure:

[0007]

[0008] Wherein,

[0009] The n1, n2, and n3 independently selected from positive integers of 1-10000;

[0010] The R1 and R2 independently selected from hydrophilic groups;

[0011] The Ar does not exist, or independently selected from electron-donating groups;

[0012] The π is selected from one of an aromatic ring, an aromatic heterocycle, a fused aromatic ring, and a fused aromatic heterocycle,

[0013] Wherein, in the aromatic ring, aromatic heterocycle, fused aromatic ring, and fused aromatic heterocycle, it contains or does not contain a chain segment with a hydrophilic group;

[0014] The hydrophilic group is selected from the following structures:

[0015]

[0016] The n4, n5, n6, n7, n8, n9, n 10 、n 11 independently selected from positive integers of 1-10000.

[0017] Furthermore, the π is selected from the following structures:

[0018]

[0019] Wherein,

[0020] The said n 12 is a positive integer selected from 1 to 10,000;

[0021] The said R3 is a hydrophilic group;

[0022] The said hydrophilic group is selected from the following structures:

[0023]

[0024] The said n4, n5, n6, n7, n8, n9, n 10 , n 11 are independently selected from positive integers of 1 to 10,000;

[0025] The said R4, R5, R6 are independently selected from one or more of a hydrogen atom, an aryl derivative, and an alkyl derivative;

[0026] All hydrogen atoms on the said alkyl derivative are unsubstituted,

[0027] or

[0028] one or more hydrogen atoms at any position on the alkyl derivative are substituted by one or more of a halogen, a hydroxyl group, an amino group, a carboxyl group, a cyano group, a nitro group, an aryl group, an alkenyl group, an alkynyl group, or an ester group;

[0029] The said aryl derivative is selected from groups containing one or more aromatic ring structures, where all hydrogen atoms on the aromatic rings are unsubstituted,

[0030] or

[0031] one or more hydrogen atoms at any position on one or more aromatic rings are substituted by one or more of a halogen, a hydroxyl group, an amino group, a carboxyl group, a cyano group, a nitro group, an aryl group, an alkenyl group, an alkynyl group, or an ester group.

[0032] Furthermore, the said electron-donating group is selected from the following structures:

[0033]

[0034] Among them,

[0035] The said R7 - R 23 are independently selected from one or more of a hydrogen atom, an aryl derivative, and an alkyl derivative;

[0036] All hydrogen atoms on the said alkyl derivative are unsubstituted,

[0037] or

[0038] One or more hydrogen atoms at any position on the alkyl derivative are substituted by one or more of a halogen, a hydroxyl group, an amino group, a carboxyl group, a cyano group, a nitro group, an aryl group, an alkenyl group, an alkynyl group or an ester group;

[0039] The aryl derivative is selected from groups containing one or more aromatic ring structures, wherein all hydrogen atoms on the aromatic rings are unsubstituted,

[0040] or

[0041] One or more hydrogen atoms at any position on one or more aromatic rings are substituted by one or more of a halogen, a hydroxyl group, an amino group, a carboxyl group, a cyano group, a nitro group, an aryl group, an alkenyl group, an alkynyl group or an ester group.

[0042] Another object of the present invention is to provide a method for preparing the above-mentioned D-A type conjugated polymer based on click chemistry, comprising the following steps:

[0043] S1. Under the protection of an inert gas, cyclopentabithiophene and a haloolefin are blended, stirred and reacted, and then purified to obtain intermediate 1;

[0044] S2. Under the protection of an inert gas, the intermediate 1, an organolithium compound and an organotin compound are blended and reacted, and then purified to obtain intermediate 2;

[0045] S3. Under the protection of an inert gas, the intermediate 2 and a monomer containing an electron-withdrawing unit are blended, a catalyst is added, and the mixture is heated and reacted, and then purified to obtain intermediate 3;

[0046] S4. Under the protection of an inert gas, the intermediate 3 and a mercapto hydrophilic compound are blended, reacted, and then purified to obtain a D-A type conjugated polymer based on click chemistry;

[0047] Wherein,

[0048] The monomer containing an electron-withdrawing unit has the following structure:

[0049]

[0050] Ar does not exist, or is independently selected from electron-donating groups;

[0051] π is selected from one of an aromatic ring, an aromatic heterocycle, a fused aromatic ring, a fused aromatic heterocycle,

[0052] wherein, among the aromatic ring, the aromatic heterocycle, the fused aromatic ring, the fused aromatic heterocycle, there is or is not a chain segment with a hydrophilic group;

[0053] The electron-donating group is selected from the following structures:

[0054]

[0055] Among them,

[0056] the R7-R 23 are independently selected from one or more of a hydrogen atom, an aryl derivative, and an alkyl derivative;

[0057] All hydrogen atoms on the alkyl derivative are unsubstituted,

[0058] or

[0059] one or more hydrogen atoms at any position on the alkyl derivative are substituted by one or more of a halogen, a hydroxyl group, an amino group, a carboxyl group, a cyano group, a nitro group, an aryl group, an alkenyl group, an alkynyl group, or an ester group;

[0060] The aryl derivative is selected from groups containing one or more aromatic ring structures, where all hydrogen atoms on the aromatic rings are unsubstituted,

[0061] or

[0062] one or more hydrogen atoms at any position on one or more aromatic rings are substituted by one or more of a halogen, a hydroxyl group, an amino group, a carboxyl group, a cyano group, a nitro group, an aryl group, an alkenyl group, an alkynyl group, or an ester group;

[0063] The mercapto hydrophilic compound contains a hydrophilic group;

[0064] The hydrophilic group is selected from the following structures:

[0065]

[0066] The n4, n5, n6, n7, n8, n9, n 10 , n 11 are independently selected from positive integers from 1 to 10000.

[0067] Furthermore, in step S3, the heating temperature is 130 - 150 °C.

[0068] Furthermore, in step S3, the catalyst is Pd(PPh3)4.

[0069] Furthermore, in step S3, the reaction needs to be carried out in an organic solvent, and the organic solvent is selected from one or more of chlorobenzene or toluene.

[0070] Furthermore, the preparation method of the monomer containing an electron-withdrawing unit includes the following steps:

[0071] L1. Under the protection of an inert gas, blend a benzothiadiazole or its derivative containing a nitro group and an aliphatic carboxylic acid, heat, add a reducing agent, react, and purify to obtain intermediate 1;

[0072] L2. Under the protection of an inert gas, blend the intermediate 1 and nitrite, stir and react, and then purify to obtain intermediate 2;

[0073] L3. Under the protection of an inert gas, blend the intermediate 2, haloolefin, and tert-butoxide, heat and react, and then purify to obtain a monomer containing an electron-withdrawing unit.

[0074] Further, in step L1, the heating temperature is 80 - 100 °C.

[0075] Further, in step L3, the heating temperature is 80 - 100 °C.

[0076] Another object of the present invention is to provide a water-soluble nanoparticle, which comprises the above click-chemistry-based D-A type conjugated polymer and is formed by dissolving it in a solvent.

[0077] Further, the particle size of the water-soluble nanoparticle is 150 - 250 nm.

[0078] Further, the mass ratio of the click-chemistry-based D-A type conjugated polymer to the solvent is 1:(1 - 2).

[0079] Another object of the present invention is to provide the application of the above water-soluble nanoparticle as a photoacoustic contrast agent in the near-infrared region.

[0080] Compared with the prior art, the main advantages of the present invention are as follows:

[0081] The present invention provides a click-chemistry-based D-A type conjugated polymer. The present invention selects a structure with strong electron-donating ability, strong electron-withdrawing ability, and a modification site. By introducing a click site at the modification site of this structure, and then using click chemistry to covalently bind a hydrophilic group to the polymer backbone, the polymer has excellent solubility and stability in aqueous solution, extends its absorption peak to the NIR-II region window, and realizes long-term stable NIR-II region PA imaging. The present invention also forms water-soluble nanoparticles by self-assembling the polymer. Through in vitro photothermal and photoacoustic experiments, the self-assembled water-soluble nanoparticles have excellent photothermal performance and photoacoustic effects, can be stored in aqueous solution for a long time without aggregation and cleavage, and have good biocompatibility, and can be applied to photoacoustic imaging in mice. Description of the Drawings

[0082] Figure 1 Shows the synthetic routes of the preparation example, Example 1, and Example 2;

[0083] Among them,

[0084] Figure 1(a) shows the synthetic route of TBZ10V in the Preparation Example;

[0085] Figure 1 (b) shows the synthetic routes of PCDT10V-SO-BBT in Example 1 and PCDT10V-SO-TBZ10V in Example 2.

[0086] Figure 2 Shows the structures of Example 1 and Example 2; wherein, n is 1 - 99 in PCDT10V-SO-BBT; n is 1 - 99 in PCDT10V-SO-TBZ10V.

[0087] Figure 3 Shows the UV-Vis-NIR absorption spectra of Application Example 1 and Application Example 2 in aqueous solution;

[0088] Wherein,

[0089] Figure 3 (a) shows the UV-Vis-NIR absorption spectrum of PCDT10V-SO-BBT@NPs in Application Example 1 in aqueous solution;

[0090] Figure 3 (b) shows the UV-Vis-NIR absorption spectrum of PCDT10V-SO-TBZ10V@NPs in Application Example 2 in aqueous solution.

[0091] Figure 4 Shows the stability of Example 1 and Example 2 in aqueous solution and PBS;

[0092] Wherein,

[0093] Figure 4 (a) shows the stability of PCDT10V-SO-BBT in Example 1 in aqueous solution and PBS;

[0094] Figure 4 (b) shows the stability of PCDT10V-SO-TBZ10V in Example 2 in aqueous solution and PBS.

[0095] Figure 5 Shows the dynamic light scattering diagrams and Zeta potential diagrams of the water-soluble nanoparticles of Application Example 1 and Application Example 2;

[0096] Wherein,

[0097] Figure 5 (a) shows the dynamic light scattering diagram of the water-soluble nanoparticle PCDT10V-SO-BBT@NPs in Application Example 1;

[0098] Figure 5(b) shows the Zeta potential diagram of the water-soluble nanoparticles PCDT10V-SO-BBT@NPs of Application Example 1;

[0099] Figure 5 (c) shows the dynamic scattering diagram of the water-soluble nanoparticles PCDT10V-SO-TBZ10V@NPs of Application Example 2;

[0100] Figure 5 (d) shows the Zeta potential diagram of the water-soluble nanoparticles PCDT10V-SO-TBZ10V@NPs of Application Example 2.

[0101] Figure 6 Shows the temperature change curves of different concentrations of Application Example 1 and Application Example 2 under 1064nm laser irradiation in the photothermal experiment;

[0102] Among them,

[0103] Figure 6 (a) shows the temperature change curves of the water-soluble nanoparticles PCDT10V-SO-BBT@NPs of Application Example 1 with different concentrations under 1064nm laser irradiation in the photothermal experiment;

[0104] Figure 6 (b) shows the temperature change curves of the water-soluble nanoparticles PCDT10V-SO-TBZ10V@NPs of Application Example 2 with different concentrations under 1064nm laser irradiation in the photothermal experiment.

[0105] Figure 7 Shows the cytotoxicity test diagrams of Application Example 1 and Application Example 2;

[0106] Among them,

[0107] Figure 7 (a) shows the cytotoxicity test diagram of the nanoparticles PCDT10V-SO-BBT@NPs of Application Example 1;

[0108] Figure 7 (b) shows the cytotoxicity test diagram of the nanoparticles PCDT10V-SO-TBZ10V@NPs of Application Example 2.

[0109] Figure 8 Shows the hemolysis test diagrams of Application Example 1 and Application Example 2;

[0110] Among them,

[0111] Figure 8 (a) shows the hemolysis test diagram of the nanoparticles PCDT10V-SO-BBT@NPs of Application Example 1;

[0112] Figure 8 (b) shows the hemolysis test chart of the nanoparticle PCDT10V-SO-TBZ10V@NPs in Application Example 2.

[0113] Figure 9 The in vitro photoacoustic test results of Application Example 1 are shown;

[0114] Among them,

[0115] Figure 9 (a) shows the photoacoustic signals at different concentrations of PCDT10V-SO-BBT@NPs under the irradiation of a Nd:YAG laser (wavelength 1064 nm) with a laser energy density of 20 mJ·cm -2 ;

[0116] Figure 9 (b) shows the photoacoustic signal intensities at different concentrations of PCDT10V-SO-BBT@NPs under the irradiation of a Nd:YAG laser (wavelength 1064 nm) with a laser energy density of 20 mJ·cm -2 ; Detailed implementation manners

[0117] To more clearly illustrate the technical solutions of the present invention, the following examples are listed. The raw materials, reactions, and post-treatment means that appear in the examples are all common raw materials on the market and technical means well-known to those skilled in the art, unless otherwise specified.

[0118] The following raw materials are used in the examples of the present invention:

[0119] BBT-C8C 10 -2Sn: 4,8-bis(5-bromo-4-(2-octyldodecyl)thienyl)-benzo[1,2-c;4,5-c']di[1,2,5]thiadiazole.

[0120] mPEG-SH: Methoxypolyethylene glycol thiol, with the product number S27339, purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0121] Preparation examples

[0122] Synthesis of the monomer TBZ10V containing an electron-withdrawing unit.

[0123] A monomer containing an electron-withdrawing unit, and its preparation method includes the following steps:

[0124] L1. Under the protection of nitrogen, add 60 mL of acetic acid and 4 g of 4,7-dibromo-5,6-dinitrobenzothiadiazole into a round-bottom flask equipped with a magnetic stirrer, heat to 100 °C, add 8 g of Fe, stir and react for 4 h. After the reaction is completed, cool to room temperature, filter, and extract the solid with ethyl acetate in a Soxhlet extractor for 12 h, and evaporate the solvent to obtain Intermediate 1;

[0125] L2. Under the protection of nitrogen, dissolve the intermediate 1 (9.33 mmol, 3 g) fully in glacial acetic acid, and take 5 mL of ultrapure water to dissolve sodium nitrite (11 mmol, 0.76 g). Add the sodium nitrite solution into the reaction flask, and stir the reaction at room temperature for 1 h. After the reaction is completed, filter by Buchner funnel, collect the ginger-yellow precipitate, and obtain the intermediate product intermediate 2;

[0126] L3. Add the intermediate 2 (0.1 mmol, 1.32 g), 10-bromo-1-decene (0.2 mmol, 1.76 g) and potassium tert-butoxide (0.42 mmol, 1.87 g) into the reaction flask, and add 5 mL of ultradry dichloromethane and 20 mL of ultradry dimethylformamide under nitrogen. Heat up to 80 °C, stir and reflux the reaction for 72 h. After monitoring by TLC that the raw materials have completely reacted, ventilate to cool down and end the reaction. Directly add the product into the silica gel column for purification, rotary evaporate the organic solvent and recrystallize to obtain the monomer (TBZ10V) containing an electron-withdrawing unit.

[0127] Example 1

[0128] A D-A type conjugated polymer based on click chemistry, and its preparation method includes the following steps:

[0129] S1. Under the protection of nitrogen, dissolve cyclopentylbithiophene (10 mmol, 1.79 g), 10-bromo-1-decene (20 mmol, 4.38 g) and potassium iodide (43.6 mg) in dimethyl sulfoxide, lower the reaction temperature to 0 °C, add potassium hydroxide (30 mmol, 1.73 g) into the reaction flask under nitrogen, and then transfer it to room temperature and stir the reaction for 48 h. After the reaction is completed, extract three times with ultrapure water and ultradry dichloromethane, collect the organic phase, dry the organic phase with MgSO4, filter and concentrate in vacuo, and purify the residue by silica gel chromatography to obtain the intermediate product 1;

[0130] S2. Under nitrogen, dissolve the intermediate product 1 (3.77 mmol, 1.72 g) in 50 mL of ultradry tetrahydrofuran, cool it to -78 °C with liquid nitrogen, and then dropwise add n-butyllithium (15.08 mmol, 6.033 mL) into the reaction flask. Then let it warm up naturally for 1 h, cool it to -78 °C again and add trimethyltin chloride tetrahydrofuran solution (1.0 M, 18.89 mL), and warm up to room temperature and react overnight. Pour the reaction mixture into water, and extract three times with dichloromethane, collect the organic phase, dry the organic phase with MgSO4, filter and concentrate in vacuo to obtain the intermediate product 2 (CDT10V);

[0131] S3. Under nitrogen, add the intermediate product 2 (0.1 mmol, 0.09 g), BBT-C8C10 - 2Sn (0.1 mmol, 0.13 g) and Pd(PPh3)4 (12 mg) were dissolved in 5 mL of ultradry chlorobenzene. The reaction was stirred and refluxed at 140 °C for 72 h. After the reaction was completed, the reaction solution was dropped into methanol for precipitation. The precipitate was collected by filtration, and the precipitate was extracted successively with methanol and n - hexane. Finally, the polymer extracted with n - hexane was rotary - evaporated to remove the organic solvent, and a small amount of tetrahydrofuran was added to dissolve it, and then it was dropped into a methanol solution for precipitation. After filtration, it was dried in vacuo to obtain the intermediate 3 (PCDT10V - BBT);

[0132] S4. The intermediate 3 (0.04 mmol, 0.05 g) and mPEG - SH (0.09 mmol, 0.44 g) were dissolved in 50 mL of ultradry dichloromethane under nitrogen conditions, and the reaction was carried out at room temperature for 48 h. After the reaction was completed, the reaction solution was transferred to a dialysis bag and dialyzed with ultrapure water for 3 days. After dialysis, the organic solvent was rotary - evaporated and freeze - dried. A D - A type conjugated polymer based on click chemistry (PCDT10V - SO - BBT) was obtained.

[0133] Example 2

[0134] A D - A type conjugated polymer based on click chemistry, and its preparation method includes the following steps:

[0135] S1. Under the protection of nitrogen, cyclopenta[2,1 - b:3,4 - b']dithiophene (10 mmol, 1.79 g), 10 - bromo - 1 - decene (20 mmol, 4.38 g) and potassium iodide (43.6 mg) were dissolved in dimethyl sulfoxide. The reaction temperature was lowered to 0 °C, and potassium hydroxide (30 mmol, 1.73 g) was added to the reaction flask under nitrogen conditions, and then it was transferred to room temperature and stirred for 48 h. After the reaction was completed, it was extracted three times with ultrapure water and ultradry dichloromethane, the organic phase was collected, the organic phase was dried with MgSO4, filtered and concentrated in vacuo, and the residue was purified by silica gel chromatography to obtain the intermediate 1;

[0136] S2. Under nitrogen conditions, the intermediate 1 (3.77 mmol, 1.72 g) was dissolved in 50 mL of ultradry tetrahydrofuran, cooled to - 78 °C with liquid nitrogen, and then n - butyllithium (15.08 mmol, 6.033 mL) was dropped into the reaction flask. Then it was allowed to warm up naturally for 1 h, cooled to - 78 °C again and trimethyltin chloride in tetrahydrofuran solution (1.0 M, 18.89 mL) was added, and the temperature was raised to room temperature and reacted overnight. The reaction mixture was poured into water and extracted three times with dichloromethane. The organic phase was collected, the organic phase was dried with MgSO4, filtered and concentrated in vacuo to obtain the intermediate 2 (CDT10V);

[0137] S3. Under nitrogen atmosphere, dissolve the monomer containing an electron-withdrawing unit prepared in the preparation example (0.1 mmol, 0.05 g), the intermediate product 2 (0.12 mmol, 0.1 g) and Pd(PPh3)4 (11.3 mg) in 5 mL of ultra-dry chlorobenzene. Stir and reflux the reaction mixture at 140 °C for 72 h. After the reaction is completed, drop the reaction solution into methanol for precipitation, and filter to collect the precipitate. The precipitate is successively extracted with methanol, n-hexane and tetrahydrofuran. Finally, the polymer obtained by extraction with tetrahydrofuran is rotary evaporated to remove the organic solvent, and a small amount of tetrahydrofuran is added to dissolve it, and then dropped into a methanol solution for precipitation. After filtration, it is dried in vacuo to obtain intermediate product 3 (PCDT10V-TBZ10V);

[0138] S4. Dissolve the intermediate product 3 (0.04 mmol, 30 mg) and mPEG-SH (0.13 mmol, 0.26 g) in 50 mL of ultra-dry dichloromethane under nitrogen atmosphere, and react at room temperature for 48 h. After the reaction is completed, transfer the reaction solution to a dialysis bag and dialyze it with ultrapure water for 3 days. After dialysis, rotary evaporate the organic solvent and freeze-dry it. A D-A type conjugated polymer based on click chemistry (PCDT10V-SO-TBZ10V) is obtained.

[0139] Figure 1 The synthetic routes of the preparation example, Example 1 and Example 2 are shown;

[0140] Among them,

[0141] Figure 1 (a) shows the synthetic route of TBZ10V in the preparation example;

[0142] Figure 1 (b) shows the synthetic routes of PCDT10V-SO-BBT in Example 1 and PCDT10V-SO-TBZ10V in Example 2.

[0143] Figure 2 The structures of Example 1 and Example 2 are shown; among them, n is 1-99 in PCDT10V-SO-BBT; n is 1-99 in PCDT10V-SO-TBZ10V.

[0144] Application Example 1

[0145] A water-soluble nanoparticle, and its preparation method includes the following steps:

[0146] Add 1 mg of PCDT10V-SO-BBT in Example 1 to 1 mL of ultrapure water, and mix well by ultrasonic to obtain water-soluble nanoparticles (PCDT10V-SO-BBT@NPs).

[0147] Application Example 2

[0148] A water-soluble nanoparticle, and its preparation method includes the following steps:

[0149] Add 1 mg of PCDT10V-SO-TBZ10V of Example 2 into 1 mL of ultrapure water, and mix evenly by ultrasonic treatment to obtain water-soluble nanoparticles (PCDT10V-SO-TBZ10V@NPs).

[0150] Test Example 1

[0151] In order to test the optical absorption ability of the D-A type conjugated polymer based on click chemistry, the ultraviolet-visible absorption spectra in the aqueous solutions of the water-soluble nanoparticles of Application Example 1 and Application Example 2 were tested.

[0152] Test method: Weigh 1 mg of Example 1 and Example 2 respectively, dissolve them in ultrapure aqueous solution, and prepare an aqueous nanoparticle solution with a concentration of 0.05 mg mL -1 At the same time, use an ultraviolet-visible spectrophotometer to perform ultraviolet-visible-near-infrared absorption spectral analysis on the finally obtained aqueous nanoparticle solution.

[0153] The results are as Figure 3 shown.

[0154] Figure 3 Shows the UV-Vis-NIR absorption spectra of Application Example 1 and Application Example 2 in aqueous solution;

[0155] Among them,

[0156] Figure 3 (a) Shows the UV-Vis-NIR absorption spectrum of PCDT10V-SO-BBT@NPs of Application Example 1 in aqueous solution;

[0157] Figure 3 (b) Shows the UV-Vis-NIR absorption spectrum of PCDT10V-SO-TBZ10V@NPs of Application Example 2 in aqueous solution.

[0158] From the above test results, it can be seen that the main absorption band range of the aqueous solution of the water-soluble nanoparticles of Application Example 1 is 750 - 1320 nm, and its peak value is located at 982 nm. The aqueous solution of the water-soluble nanoparticles of Application Example 2 has an absorption band centered at 975 nm in the near-infrared window. The absorption bands of the aqueous solutions of the water-soluble nanoparticles of Application Example 1 and Application Example 2 can extend to the second near-infrared window.

[0159] Test Example 2

[0160] Test the stability of PCDT10V-SO-BBT of Example 1 and PCDT10V-SO-TBZ10V of Example 2 in aqueous solution and PBS solution.

[0161] Test method: Dissolve PCDT10V-SO-BBT of Example 1 and PCDT10V-SO-TBZ10V of Example 2 in ultrapure water and PBS solution respectively to prepare a test solution with a concentration of 0.05 mg / mL. Measure the particle size every 3 days using a nanoparticle size analyzer for 15 days.

[0162] The test results are as Figure 4 shown.

[0163] Figure 4 shows the stability of Example 1 and Example 2 in aqueous solution and PBS;

[0164] Among them,

[0165] Figure 4 (a) shows the stability of PCDT10V-SO-BBT of Example 1 in aqueous solution and PBS;

[0166] Figure 4 (b) shows the stability of PCDT10V-SO-TBZ10V of Example 2 in aqueous solution and PBS.

[0167] From the above test results, it can be seen that the particle size of the polymers in Examples 1-2 did not change significantly within 15 days, and the solution still maintained its initial clear transparency and color.

[0168] It can be seen that the polymers of the present invention have good solubility and stability in different media and have the potential to be used as photoacoustic contrast agents in the near-infrared region.

[0169] Test Example 3

[0170] Test the size and Zeta potential of the aqueous solutions of the water-soluble nanoparticles of Application Example 1 and Application Example 2.

[0171] Test method: Weigh 1 mg of Example 1 and Example 2 respectively, dissolve them in ultrapure aqueous solution to prepare an aqueous solution of nanoparticles with a concentration of 0.05 mg mL -1 . Put the prepared aqueous solutions of nanoparticles into the cell respectively, and use Nano-ZS90 to measure the size and Zeta potential of the aqueous solutions of nanoparticles.

[0172] The test results are as Figure 5 shown.

[0173] Figure 5 shows the dynamic scattering diagram and Zeta potential diagram of the water-soluble nanoparticles of Application Example 1 and Application Example 2;

[0174] Among them,

[0175] Figure 5 (a) shows the dynamic scattering diagram of the water-soluble nanoparticles PCDT10V-SO-BBT@NPs of Application Example 1;

[0176] Figure 5 (b) shows the Zeta potential diagram of the water-soluble nanoparticles PCDT10V-SO-BBT@NPs of Application Example 1;

[0177] Figure 5 (c) shows the dynamic scattering diagram of the water-soluble nanoparticles PCDT10V-SO-TBZ10V@NPs of Application Example 2;

[0178] Figure 5 (d) shows the Zeta potential diagram of the water-soluble nanoparticles PCDT10V-SO-TBZ10V@NPs of Application Example 2.

[0179] From the above test results, it can be seen that the particle size ranges of the water-soluble nanoparticles in Application Example 1 and Application Example 2 are 150 - 250 nm, and the Zeta potentials are -12.1 mV and -34.9 mV respectively. The experimental data show that Application Example 1 and Application Example 2 have excellent stability, which is beneficial to long-term blood circulation in the body, indicating that the water-soluble nanoparticles in Application Example 1 and Application Example 2 have the potential to be used as photoacoustic contrast agents in the near-infrared region.

[0180] Test Example 4

[0181] In view of the strong absorption of Application Example 1 and Application Example 2 in the near-infrared window region, the photothermal properties of Application Example 1 and Application Example 2 were tested through photothermal experiments.

[0182] Test method: Weigh the polymers of Example 1 and Example 2 respectively, add them into ultrapure water, and dissolve them thoroughly by ultrasonic treatment to prepare aqueous nanoparticle solutions of 200 μg mL -1 and 400 μg mL -1 . Add them into a 600 μL centrifuge tube, fix the positions of the centrifuge tube and the laser, and then irradiate the centrifuge tube with a 1064 nm laser (2 W cm -2 ) for 10 min and then turn off the laser and cool it to room temperature. During the whole process, record the temperature of the solution every 30 s. Then, the photothermal conversion efficiency of CDT10V-SO-BBT and CDT10V-SO-TBZ under the irradiation of a 1064 nm laser (1 W cm -2 ) was calculated. Finally, the photothermal conversion efficiency (PCE) was calculated by formula (1):

[0183]

[0184] where h and S are the heat transfer coefficient and the surface area of the container respectively. T Max and TSur are the highest steady-state temperature and the ambient temperature, respectively. Q Dis is the heat dissipated by the laser mediated by the solvent and the container. I is the laser power; A is the light absorption intensity of the sample at λ. The value of S is calculated by formula (2):

[0185]

[0186] where m and C p are the mass (0.6 g) and heat capacity (4.2 J / g) of water, and τ s is the time constant of the system heat transfer and can be obtained from formula (3):

[0187] t = τ s × (lnθ) (3)

[0188] where t is the time of the cooling process, and θ is a dimensionless parameter based on temperature and can be determined by formula (4):

[0189]

[0190] where t is the temperature during the cooling process.

[0191] The test results are as Figure 6 shown.

[0192] Figure 6 show the temperature change curves of Application Example 1 and Application Example 2 with different concentrations under 1064 nm laser irradiation in the photothermal experiment;

[0193] where

[0194] Figure 6 (a) shows the temperature change curves of the water-soluble nanoparticles PCDT10V-SO-BBT@NPs of Application Example 1 with different concentrations under 1064 nm laser irradiation in the photothermal experiment; where the label H2O in the figure represents an ultrapure aqueous solution;

[0195] Figure 6 (b) shows the temperature change curves of the water-soluble nanoparticles PCDT10V-SO-TBZ10V@NPs of Application Example 2 with different concentrations under 1064 nm laser irradiation in the photothermal experiment; where the label H2O in the figure represents an ultrapure aqueous solution.

[0196] As Figure 6 shown, after 10 min of irradiation with NIR-II laser (1064 nm, 2 W cm -2 ), the temperatures of the aqueous solutions of Application Example 1 and Application Example 2 gradually increased, while the temperature of the ultrapure aqueous solution group did not change significantly. Application Example 1 and Application Example 2 at 400 μg mL -1At the concentrations of, the temperatures can reach 65.3 °C and 64.8 °C respectively, which is sufficient to cause irreversible thermal damage to tumors. And in the five cycles of on / off treatment of the laser (1064 nm, 2 W cm -2 ), there is no obvious change in the rising and cooling trends of the solution temperature. These data indicate that Application Example 1 and Application Example 2 have excellent stability and high photothermal conversion efficiency for tumor photothermal therapy.

[0197] Test Example 5

[0198] Biocompatibility is crucial for PA imaging. Therefore, before applying Application Example 1 and Application Example 2 to in vivo PA imaging, their biocompatibility was evaluated through in vitro cytotoxicity experiments and hemolysis experiments.

[0199] Test method:

[0200] (1) In vitro cytotoxicity experiment: In the in vitro cytotoxicity experiment, A549 and HepG2 cells (gifted by Gannan Medical University, the cells are prior art) were treated with different concentrations of PCDT10V-SO-BBT@NPs for 24 h, and cell viability was evaluated by the MTT method. Meanwhile, HepG2 and AML-12 cells (gifted by Gannan Medical University, the cells are prior art) were treated with different concentrations of PCDT10V-SO-TBZ10V@NPs for 24 h, and cell viability was evaluated by the MTT method.

[0201] (2) Hemolysis experiment: After incubating different concentrations of Application Example 1 and Application Example 2 with 4% red blood cell suspension for 1 h, the hemolysis rate was measured.

[0202] The test results are as Figure 7 and Figure 8 shown.

[0203] Figure 7 Shows the cytotoxicity test charts of Application Example 1 and Application Example 2;

[0204] Among them,

[0205] Figure 7 (a) Shows the cytotoxicity test chart of the nanoparticles PCDT10V-SO-BBT@NPs of Application Example 1;

[0206] Figure 7 (b) Shows the cytotoxicity test chart of the nanoparticles PCDT10V-SO-TBZ10V@NPs of Application Example 2.

[0207] Figure 8 Shows the hemolysis test charts of Application Example 1 and Application Example 2;

[0208] Among them,

[0209] Figure 8 (a) shows the hemolysis test diagram of the nanoparticles PCDT10V-SO-BBT@NPs in Application Example 1;

[0210] Figure 8 (b) shows the hemolysis test diagram of the nanoparticles PCDT10V-SO-TBZ10V@NPs in Application Example 2.

[0211] The results of the in vitro cytotoxicity experiment in Application Example 1 are as Figure 7 (a) shown. After culturing for 24 h, in the concentration range of 0 - 160 μg mL -1 , as the concentration of PCDT10V-SO-BBT@NPs increased, the cell viability gradually decreased. However, even at a concentration of 160 μg·mL -1 , the survival rate of HeLa cells was still over 90%. The results of the in vitro cytotoxicity experiment in Application Example 2 are as Figure 7 (b) shown. In the concentration range of 0 - 160 μg mL -1 , both types of cells could maintain good activity. Even at a concentration of 160 μg mL -1 , the survival rates of Hep G2 cells and AML-12 cells were still over 90%.

[0212] Subsequently, the hemolytic properties of Application Example 1 and Application Example 2 were further evaluated through hemolysis experiments, which are a prerequisite for in vivo applications. The results of the hemolysis experiment are as Figure 8 shown. Even at a concentration of 160 μg mL -1 , no obvious erythrocyte lysis was observed, and the hemolysis rate was still less than 5%.

[0213] In summary, the results of the in vitro cytotoxicity experiment and hemolysis experiment indicate that the PCDT10V-SO-BBT@NPs in Application Example 1 and the PCDT10V-SO-TBZ10V@NPs in Application Example 2 have good biocompatibility and broad application prospects in in vivo PA imaging.

[0214] Test Example 6

[0215] In view of the good biocompatibility and photothermal properties of Application Example 1, its feasibility as an in vivo imaging contrast agent was further evaluated through in vitro photoacoustic tests. A solid model (containing 2 wt% agar, 1 wt% internal lipid, and the balance water) was used to simulate in vitro conditions, and then the PA signal intensities of the water-soluble nanoparticles of Application Example 1 at different concentrations were measured.

[0216] Test method: In vitro experiments, a solid model containing agar (2 wt%), internal lipid (1 wt%), and the balance water was first prepared. Subsequently, 1 mg of Example 1 was weighed and dissolved in ultrapure water to prepare an aqueous solution of nanoparticles (100 μL × 1 mg mL -1 ). It was injected into a transparent quartz tube with a diameter of 0.9 mm, and then both ends of the quartz tube were sealed and embedded in the solid model.

[0217] Subsequently, the solid model was placed on the experimental platform and detected with a PA imaging system under laser irradiation with an energy density of 20 mJ·cm -2 . The scanning length was 10 mm, the scanning step was 100 μm, and PA images were obtained.

[0218] The test results are as Figure 9 shown.

[0219] Figure 9 The in vitro photoacoustic test results of Application Example 1 are shown;

[0220] Among them,

[0221] Figure 9 (a) shows the photoacoustic signals at different concentrations of PCDT10V-SO-BBT@NPs under Nd:YAG laser irradiation (wavelength 1064 nm) with an energy density of 20 mJ·cm -2 .

[0222] Figure 9 (b) shows the photoacoustic signal intensities at different concentrations of PCDT10V-SO-BBT@NPs under Nd:YAG laser irradiation (wavelength 1064 nm) with an energy density of 20 mJ·cm -2 .

[0223] It can be seen from Figure 9 (a) and 9(b) that as the concentration of the nanoparticle solution increases from 0.5 mg·mL -1 to 2 mg·mL -1 , the PA signal-to-noise ratio of PCDT10V-SO-BBT@NPs increases. In addition, it was also observed that the imaging signal was almost positively correlated with the concentration of PCDT10V-SO-BBT@NPs. And at a concentration of 2 mg·mL -1 , it showed the strongest PA signal-to-noise ratio and had the ability of in vivo PA imaging.

[0224] It is obvious to those skilled in the art that the present invention is not limited to the details of the above-described exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, in any regard, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention.

[0225] In addition, it should be understood that although this specification is described in terms of embodiments, not every embodiment only contains an independent technical solution. This narrative manner of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A D-A type conjugated polymer based on click chemistry, characterized in that The D-A type conjugated polymer based on click chemistry has the following structure: Wherein, The n1, n2, and n3 are independently selected from positive integers of 1 to 10,000; The R1 and R2 are independently selected from hydrophilic groups; The Ar does not exist, or is independently selected from electron-donating groups; The π is selected from one of an aromatic ring, a heteroaromatic ring, a fused aromatic ring, and a fused heteroaromatic ring, Wherein, in the aromatic ring, heteroaromatic ring, fused aromatic ring, and fused heteroaromatic ring, there is or is not a segment with a hydrophilic group; The hydrophilic group is selected from the following structures: The n4, n5, n6, n7, n8, n9, n 10 , n 11 are independently selected from positive integers of 1 to 10000.

2. The D-A type conjugated polymer based on click chemistry according to claim 1, characterized in that, The π is selected from the following structures: Wherein, Said n 12 A positive integer selected from 1 to 10000; The R3 is a hydrophilic group; The hydrophilic group is selected from the following structures: Said n4, n5, n6, n7, n8, n9, n 10 , n 11 are independently selected from positive integers of 1 to 10,000; The R4, R5, and R6 are independently selected from one or more of a hydrogen atom, an aryl derivative, and an alkyl derivative; All hydrogen atoms on the alkyl derivative are unsubstituted, Or One or more hydrogen atoms at any position on the alkyl derivative are substituted by one or more of a halogen, a hydroxyl group, an amino group, a carboxyl group, a cyano group, a nitro group, an aryl group, an alkenyl group, an alkynyl group, or an ester group; The aryl derivative is selected from a group containing one or more aromatic ring structures, wherein all hydrogen atoms on the aromatic rings are unsubstituted, Or One or more hydrogen atoms at any position on one or more aromatic rings are substituted by one or more of a halogen, a hydroxyl group, an amino group, a carboxyl group, a cyano group, a nitro group, an aryl group, an alkenyl group, an alkynyl group, or an ester group.

3. The D-A type conjugated polymer based on click chemistry according to claim 1, characterized in that, The electron-donating group is selected from the following structures: Wherein, The R7-R 23 are independently selected from one or more of a hydrogen atom, an aryl derivative, and an alkyl derivative; All hydrogen atoms on the alkyl derivative are unsubstituted, Or One or more hydrogen atoms at any position on the alkyl derivative are substituted by one or more of a halogen, a hydroxyl group, an amino group, a carboxyl group, a cyano group, a nitro group, an aryl group, an alkenyl group, an alkynyl group, or an ester group; The aryl derivative is selected from a group containing one or more aromatic ring structures, wherein all hydrogen atoms on the aromatic rings are unsubstituted, Or One or more hydrogen atoms at any position on one or more aromatic rings are substituted by one or more of a halogen, a hydroxyl group, an amino group, a carboxyl group, a cyano group, a nitro group, an aryl group, an alkenyl group, an alkynyl group, or an ester group.

4. The preparation method of the D-A type conjugated polymer based on click chemistry according to any one of claims 1-3, characterized in that, Including the following steps: S1. Under the protection of an inert gas, cyclopentabithiophene and haloolefin are blended, stirred and reacted, and purified to obtain intermediate 1; S2. Under the protection of an inert gas, the intermediate 1, an organolithium compound, and an organotin compound are blended and reacted, and purified to obtain intermediate 2; S3. Under the protection of an inert gas, the intermediate 2 and a monomer containing an electron-withdrawing unit are blended, a catalyst is added, heated and reacted, and purified to obtain intermediate 3; S4. Under the protection of an inert gas, the intermediate 3 and a mercapto hydrophilic compound are blended, reacted, and purified to obtain a D-A type conjugated polymer based on click chemistry; Wherein, The monomer containing an electron-withdrawing unit has the following structure: The Ar does not exist, or is independently selected from electron-donating groups; The π is selected from one of an aromatic ring, a heteroaromatic ring, a fused aromatic ring, and a fused heteroaromatic ring, Wherein, in the aromatic ring, heteroaromatic ring, fused aromatic ring, and fused heteroaromatic ring, there is or is not a segment with a hydrophilic group; The electron-donating group is selected from the following structures: Wherein, The R7-R 23 are independently selected from one or more of a hydrogen atom, an aryl derivative, and an alkyl derivative; All hydrogen atoms on the alkyl derivative are unsubstituted. Or One or more hydrogen atoms at any position on the alkyl derivative are substituted by one or more of a halogen, a hydroxyl group, an amino group, a carboxyl group, a cyano group, a nitro group, an aryl group, an alkenyl group, an alkynyl group or an ester group; The aryl derivative is selected from groups containing one or more aromatic ring structures, where all hydrogen atoms on the aromatic rings are unsubstituted. Or One or more hydrogen atoms at any position on one or more aromatic rings are substituted by one or more of a halogen, a hydroxyl group, an amino group, a carboxyl group, a cyano group, a nitro group, an aryl group, an alkenyl group, an alkynyl group or an ester group; The mercapto hydrophilic compound contains a hydrophilic group. The hydrophilic group is selected from the following structures: Said n4, n5, n6, n7, n8, n9, n 10 , n 11 are independently selected from positive integers of 1 to 10,000.

5. The preparation method of the D-A type conjugated polymer based on click chemistry according to claim 4, wherein In step S3, the heating temperature is 130 - 150 °C.

6. The preparation method of the D-A type conjugated polymer based on click chemistry according to claim 4, characterized in that The preparation method of the monomer containing an electron-withdrawing unit includes the following steps: L1. Under the protection of an inert gas, blend a benzothiadiazole or its derivative containing a nitro group and an aliphatic carboxylic acid, heat, add a reducing agent, react, and purify to obtain intermediate 1; L2. Under the protection of an inert gas, blend the intermediate 1 and a nitrite, stir and react, and purify to obtain intermediate 2; L3. Under the protection of an inert gas, blend the intermediate 2, a haloolefin and a tert-butoxide, heat and react, and purify to obtain a monomer containing an electron-withdrawing unit.

7. A water-soluble nanoparticle, characterized in that, The water-soluble nanoparticles include the click-chemistry-based D-A type conjugated polymer as described in any one of claims 1 - 3, and are formed by dissolving it in a solvent.

8. The water-soluble nanoparticles according to claim 7, wherein, The particle size of the water-soluble nanoparticles is 150 - 250 nm.

9. The water-soluble nanoparticles according to claim 7, characterized in that, The mass ratio of the click-chemistry-based D-A type conjugated polymer to the solvent is 1:(1 - 2).

10. Use of the water-soluble nanoparticles as claimed in claim 7 as a photoacoustic contrast agent in the near-infrared region.

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