A click chemistry-based DA-type conjugated polymer, its preparation method and application

Abstract

This invention provides a D-A type conjugated polymer based on click chemistry, its preparation method, and its applications. The invention selects a structure with strong electron-donating and electron-deficient capabilities and containing modification sites. By introducing click sites into these modification sites, click chemistry is used to covalently bond hydrophilic groups to the polymer backbone, resulting in excellent solubility and stability of the polymer in aqueous solution. This extends the absorption peak to the NIR-II region window, achieving long-term stable NIR-II region PA imaging. Furthermore, the invention allows the polymer to self-assemble into water-soluble nanoparticles. In vitro photothermal and photoacoustic experiments show that the self-assembled water-soluble nanoparticles exhibit excellent photothermal and photoacoustic properties, can be stored in aqueous solution for extended periods without aggregation or fragmentation, and possess good biocompatibility, making them suitable for in vivo photoacoustic imaging in mice.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a DA-type conjugated polymer based on click chemistry, its preparation method, and its applications. Background Technology

[0002] Photoacoustic imaging (PAI) is a composite imaging technique combining photoexcitation and ultrasound detection, which effectively facilitates in vivo imaging. It boasts advantages such as excellent tissue penetration, high image contrast, and precise spatial resolution. In particular, when using a near-infrared II (NIR-II, 1000-1700nm) excitation source for PAI, the lower absorption and scattering of light in this wavelength band significantly improves the signal-to-noise ratio and enables deeper imaging penetration, thereby greatly enhancing image quality. Based on these advantages, NIR-II window photoacoustic imaging technology holds promise for clinical applications in tumor detection and cardiovascular disease imaging, demonstrating broad application prospects.

[0003] Semiconductor polymers have shown great potential in the biomedical field due to their unique optical properties and ease of modification. However, most of these polymers are insoluble in water due to their skeletal structure, often requiring post-modification. For example, semiconductor polymers can be encapsulated into amphiphilic polymers through microemulsions or nanoprecipitations to prepare water-dispersible nanoparticles, thereby achieving their solubility in aqueous solutions, long-term stability, and bioapplication. This method has made significant progress in recent decades. However, this preparation method has its limitations. Literature reports that amphiphilic copolymers leak from nanoparticles, accumulating in the bloodstream and ultimately leading to poor imaging results and biotoxicity. Furthermore, interactions between nanoparticles and proteins or other substances in the body accelerate nanoparticle disintegration. This change can alter their optical properties and impair their biodistribution.

[0004] To overcome these potential drawbacks and improve their in vivo performance, many other methods have been successfully used to prepare nanoparticles, such as solution self-assembly and microfluidic methods. However, designing stable, size-consistent, and batch-to-batch-variety optical imaging nanoparticles remains a major challenge. To overcome the poor solubility of semiconductor polymers in solvents such as aqueous solutions and thus break through their limitations in in vivo bioimaging applications, a new technological solution is urgently needed to address the problems of existing technologies. Summary of the Invention

[0005] Based on this, the present invention provides a click chemistry-based DA-type conjugated polymer, its preparation method, and its applications. The present invention selects a structure with strong electron-donating and electron-deficient capabilities and containing modification sites. By introducing click sites into these modification sites, click chemistry is used to covalently bond hydrophilic groups to the polymer backbone, resulting in excellent solubility and stability of the polymer in aqueous solution. This extends the absorption peak to the NIR-II region window, achieving long-term stable NIR-II region PA imaging. The present invention also allows the polymer to self-assemble into water-soluble nanoparticles. In vitro photothermal and photoacoustic experiments show that the self-assembled water-soluble nanoparticles exhibit excellent photothermal and photoacoustic properties, can be stored in aqueous solution for extended periods without aggregation or fragmentation, and possess good biocompatibility, making them suitable for in vivo photoacoustic imaging in mice.

[0006] One object of the present invention is to provide a click chemistry-based DA-type conjugated polymer having the following structure:

[0007]

[0008] in,

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

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

[0011] The Ar is absent, or is independently selected from electron-donating groups;

[0012] The π is selected from one of aromatic rings, aromatic heterocyclic rings, fused aromatic rings, and fused aromatic heterocyclic rings.

[0013] The aromatic ring, aromatic heterocyclic ring, fused aromatic ring, and fused aromatic heterocyclic ring may or may not contain segments with hydrophilic groups.

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

[0015]

[0016] The n4, n5, n6, n7, n8, n9, n 10 n 11 Positive integers selected independently from 1 to 10000.

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

[0018]

[0019] in,

[0020] The n 12 Positive integers selected from 1 to 10000;

[0021] R3 is a hydrophilic group;

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

[0023]

[0024] The n4, n5, n6, n7, n8, n9, n 10 n 11 Positive integers independently selected from 1 to 10000;

[0025] R4, R5, and R6 are independently selected from one or more of hydrogen atoms, aryl derivatives, and alkyl derivatives;

[0026] All hydrogen atoms on the alkyl derivative were not substituted.

[0027] or

[0028] One or more hydrogen atoms at any position on an alkyl derivative are substituted by one or more halogen, hydroxyl, amino, carboxyl, cyano, nitro, aryl, olefinic, alkyneic, or ester groups.

[0029] The aryl derivative is selected from groups containing one or more aromatic ring structures, wherein all hydrogen atoms on the aromatic ring 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 halogen, hydroxyl, amino, carboxyl, cyano, nitro, aryl, olefinic, alkyneic, or ester groups.

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

[0033]

[0034] in,

[0035] The R7-R 23 Independently selected from one or more of hydrogen atoms, aryl derivatives, and alkyl derivatives;

[0036] All hydrogen atoms on the alkyl derivative were not substituted.

[0037] or

[0038] One or more hydrogen atoms at any position on an alkyl derivative are substituted by one or more halogen, hydroxyl, amino, carboxyl, cyano, nitro, aryl, olefinic, alkyneic, or ester groups.

[0039] The aryl derivative is selected from groups containing one or more aromatic ring structures, wherein all hydrogen atoms on the aromatic ring 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 halogen, hydroxyl, amino, carboxyl, cyano, nitro, aryl, olefinic, alkyneic, or ester groups.

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

[0043] S1. Under the protection of an inert gas, cyclopentylthiophene and haloalkene were blended, stirred, and purified to obtain intermediate product 1.

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

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

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

[0047] in,

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

[0049]

[0050] The Ar is absent, or is independently selected from electron-donating groups;

[0051] The π is selected from one of aromatic rings, aromatic heterocyclic rings, fused aromatic rings, and fused aromatic heterocyclic rings.

[0052] The aromatic ring, aromatic heterocyclic ring, fused aromatic ring, and fused aromatic heterocyclic ring may or may not contain segments with hydrophilic groups.

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

[0054]

[0055] in,

[0056] The R7-R 23 Independently selected from one or more of hydrogen atoms, aryl derivatives, and alkyl derivatives;

[0057] All hydrogen atoms on the alkyl derivative were not substituted.

[0058] or

[0059] One or more hydrogen atoms at any position on an alkyl derivative are substituted by one or more halogen, hydroxyl, amino, carboxyl, cyano, nitro, aryl, olefinic, alkyneic, or ester groups.

[0060] The aryl derivative is selected from groups containing one or more aromatic ring structures, wherein all hydrogen atoms on the aromatic ring 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 halogen, hydroxyl, amino, carboxyl, cyano, nitro, aryl, olefinic, alkyneic, or ester groups;

[0063] The thiol 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 Positive integers selected independently 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, which is selected from one or more of chlorobenzene or toluene.

[0070] Furthermore, the method for preparing the monomer containing the electron-withdrawing unit includes the following steps:

[0071] L1. Under the protection of an inert gas, benzothiadiazole or its derivative containing a nitro group is blended with an aliphatic carboxylic acid, heated, a reducing agent is added, the reaction is carried out, and the mixture is purified to obtain intermediate 1.

[0072] L2. Under the protection of an inert gas, intermediate 1 and nitrite are mixed, stirred and reacted, and purified to obtain intermediate 2.

[0073] L3. Under the protection of an inert gas, intermediate 2, haloolefin and tert-butyl alkoxide are blended, heated and reacted, and purified to obtain a monomer containing an electron-withdrawing unit.

[0074] Furthermore, in step L1, the heating temperature is 80-100℃.

[0075] Furthermore, in step L3, the heating temperature is 80-100℃.

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

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

[0078] Furthermore, the mass ratio of the click chemistry-based DA-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-mentioned water-soluble nanoparticles 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] This invention provides a DA-type conjugated polymer based on click chemistry. The invention selects a structure with strong electron-donating and electron-deficient capabilities and containing modification sites. By introducing click sites into these modification sites, click chemistry is used to covalently bond hydrophilic groups to the polymer backbone, resulting in excellent solubility and stability of the polymer in aqueous solution. This extends the absorption peak to the NIR-II region window, achieving long-term stable NIR-II region PA imaging. Furthermore, this invention allows the polymer to self-assemble into water-soluble nanoparticles. In vitro photothermal and photoacoustic experiments show that the self-assembled water-soluble nanoparticles exhibit excellent photothermal and photoacoustic properties, can be stored in aqueous solution for extended periods without aggregation or fragmentation, and possess good biocompatibility, making them suitable for in vivo photoacoustic imaging in mice. Attached Figure Description

[0082] Figure 1 The synthetic routes of the preparation examples, Example 1, and Example 2 are shown;

[0083] in,

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

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

[0086] Figure 2 The structures of Embodiment 1 and Embodiment 2 are shown; wherein, n is 1-99 in PCDT10V-SO-BBT and n is 1-99 in PCDT10V-SO-TBZ10V.

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

[0088] in,

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

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

[0091] Figure 4 The stability of Examples 1 and 2 in aqueous solution and PBS is shown;

[0092] in,

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

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

[0095] Figure 5 Dynamic scattering patterns and Zeta potential diagrams of water-soluble nanoparticles in Application Examples 1 and 2 are shown.

[0096] in,

[0097] Figure 5 (a) shows the dynamic scattering pattern of the water-soluble nanoparticles 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 in Application Example 1;

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

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

[0101] Figure 6 The temperature change curves of Application Example 1 and Application Example 2 with different concentrations of 1064nm laser irradiation are shown in the photothermal experiment.

[0102] in,

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

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

[0105] Figure 7 The cytotoxicity test results for Application Example 1 and Application Example 2 are shown.

[0106] in,

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

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

[0109] Figure 8 The hemolysis test diagrams for Application Example 1 and Application Example 2 are shown;

[0110] in,

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

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

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

[0114] in,

[0115] Figure 9 (a) shows that PCDT10V-SO-BBT@NPs can operate at a laser energy density of 20 mJ·cm⁻¹. -2 Photoacoustic signals at different concentrations under Nd:YAG laser irradiation (wavelength 1064nm);

[0116] Figure 9 (b) This demonstrates the performance of PCDT10V-SO-BBT@NPs at a laser energy density of 20 mJ·cm⁻¹. -2 The photoacoustic signal intensity at different concentrations under Nd:YAG laser irradiation (wavelength 1064nm). Detailed Implementation

[0117] To more clearly illustrate the technical solution of the present invention, the following embodiments are provided. Unless otherwise stated, the raw materials, reactions, and post-processing methods appearing in the embodiments are all commercially available raw materials and technical methods well known to those skilled in the art.

[0118] The following raw materials are used in the embodiments 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']bis[1,2,5]thiadiazole.

[0120] mPEG-SH: Methoxy polyethylene glycol thiol, brand name S27339, purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0121] Preparation Example

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

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

[0124] L1. Under nitrogen protection, 60 mL of acetic acid and 4 g of 4,7-dibromo-5,6-dinitrobenzothiadiazole were added to a round-bottom flask equipped with a stir bar. The mixture was heated to 100 °C, and 8 g of Fe was added. The mixture was stirred for 4 h. After the reaction was completed, the mixture was cooled to room temperature, filtered, and the solid was extracted with ethyl acetate using a Soxhlet extraction method for 12 h. The solvent was evaporated to obtain intermediate 1.

[0125] L2. Under nitrogen protection, intermediate 1 (9.33 mmol, 3 g) was completely dissolved in glacial acetic acid, and sodium nitrite (11 mmol, 0.76 g) was dissolved in 5 mL of ultrapure water. The sodium nitrite solution was added to the reaction flask, and the reaction was stirred at room temperature for 1 h. After the reaction was completed, the precipitate was collected by suction filtration through a Buchner funnel to obtain intermediate 2.

[0126] L3. 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) were added to a reaction flask. Under nitrogen atmosphere, 5 mL of ultra-dry dichloromethane and 20 mL of ultra-dry dimethylformamide were added. The mixture was heated to 80 °C and refluxed with stirring for 72 h. After the starting materials were completely reacted, the reaction was stopped by purging and cooling. The product was directly added to a silica gel column for purification. The organic solvent was then evaporated by rotary evaporation and recrystallized to obtain the monomer containing the electron-withdrawing unit (TBZ10V).

[0127] Example 1

[0128] A click chemistry-based DA-type conjugated polymer is prepared by the following steps:

[0129] S1. Under nitrogen protection, cyclopentathiophene (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. The mixture was then transferred to room temperature and stirred for 48 h. After the reaction was completed, the mixture was extracted three times with ultrapure water and ultra-dry dichloromethane. The organic phase was collected, dried over MgSO4, filtered, and concentrated under vacuum. The residue was purified by silica gel chromatography to obtain intermediate product 1.

[0130] S2. Under nitrogen atmosphere, intermediate 1 (3.77 mmol, 1.72 g) was dissolved in 50 mL of ultra-dry tetrahydrofuran, cooled to -78 °C with liquid nitrogen, and then n-butyllithium (15.08 mmol, 6.033 mL) was added dropwise to the reaction flask. The mixture was then naturally heated for 1 h, cooled again to -78 °C, and a trimethyltin chloride tetrahydrofuran solution (1.0 M, 18.89 mL) was added. The mixture was heated to room temperature and reacted overnight. The reaction mixture was poured into water and extracted three times with dichloromethane. The organic phase was collected, dried over MgSO4, filtered, and concentrated under vacuum to give intermediate 2 (CDT10V).

[0131] S3. Under nitrogen atmosphere, the intermediate product 2 (0.1 mmol, 0.09 g) and BBT-C8C were mixed.10 -2Sn (0.1 mmol, 0.13 g) and Pd(PPh3)4 (12 mg) were dissolved in 5 mL of ultra-dry chlorobenzene. The mixture was stirred and refluxed at 140 °C for 72 h. After the reaction was completed, the reaction solution was added dropwise to methanol to precipitate the product. The precipitate was collected by filtration and extracted with methanol and n-hexane sequentially. Finally, the polymer extracted with n-hexane was rotary evaporated to obtain organic solvent, and a small amount of tetrahydrofuran was added to dissolve it. The polymer was then added dropwise to methanol solution to precipitate the product. After filtration, the product was dried under vacuum to obtain intermediate product 3 (PCDT10V-BBT).

[0132] S4. The intermediate product 3 (0.04 mmol, 0.05 g) and mPEG-SH (0.09 mmol, 0.44 g) were dissolved in 50 mL of ultra-dry dichloromethane under nitrogen atmosphere and reacted 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 the solution was lyophilized. A click chemistry-based DA-type conjugated polymer (PCDT10V-SO-BBT) was obtained.

[0133] Example 2

[0134] A click chemistry-based DA-type conjugated polymer is prepared by the following steps:

[0135] S1. Under nitrogen protection, cyclopentathiophene (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. The mixture was then transferred to room temperature and stirred for 48 h. After the reaction was completed, the mixture was extracted three times with ultrapure water and ultra-dry dichloromethane. The organic phase was collected, dried over MgSO4, filtered, and concentrated under vacuum. The residue was purified by silica gel chromatography to obtain intermediate product 1.

[0136] S2. Under nitrogen atmosphere, intermediate 1 (3.77 mmol, 1.72 g) was dissolved in 50 mL of ultra-dry tetrahydrofuran, cooled to -78 °C with liquid nitrogen, and then n-butyllithium (15.08 mmol, 6.033 mL) was added dropwise to the reaction flask. The mixture was then naturally heated for 1 h, cooled again to -78 °C, and a trimethyltin chloride tetrahydrofuran solution (1.0 M, 18.89 mL) was added. The mixture was heated to room temperature and reacted overnight. The reaction mixture was poured into water and extracted three times with dichloromethane. The organic phase was collected, dried over MgSO4, filtered, and concentrated under vacuum to obtain intermediate 2 (CDT10V).

[0137] S3. Under nitrogen atmosphere, the monomer containing the 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) were dissolved in 5 mL of ultra-dry chlorobenzene. The mixture was stirred and refluxed at 140 °C for 72 h. After the reaction was completed, the reaction solution was added dropwise to methanol to precipitate the product, and the precipitate was collected by filtration. The precipitate was extracted sequentially with methanol, n-hexane, and tetrahydrofuran. Finally, the polymer extracted with tetrahydrofuran was rotary evaporated to obtain organic solvent, and a small amount of tetrahydrofuran was added to dissolve it before being added dropwise to methanol solution to precipitate the product. After filtration, the product was dried under vacuum to obtain intermediate product 3 (PCDT10V-TBZ10V).

[0138] S4. The intermediate product 3 (0.04 mmol, 30 mg) and mPEG-SH (0.13 mmol, 0.26 g) were dissolved in 50 mL of ultra-dry dichloromethane under nitrogen atmosphere and reacted 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 the solution was lyophilized. A click chemistry-based DA-type conjugated polymer (PCDT10V-SO-TBZ10V) was obtained.

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

[0140] in,

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

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

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

[0144] Application Example 1

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

[0146] 1 mg of PCDT10V-SO-BBT from Example 1 was added to 1 mL of ultrapure water and mixed by ultrasonication to obtain water-soluble nanoparticles (PCDT10V-SO-BBT@NPs).

[0147] Application Example 2

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

[0149] 1 mg of PCDT10V-SO-TBZ10V from Example 2 was added to 1 mL of ultrapure water and mixed by ultrasonication to obtain water-soluble nanoparticles (PCDT10V-SO-TBZ10V@NPs).

[0150] Test Example 1

[0151] To test the optical absorption capability of the click chemistry-based DA-type conjugated polymer, the UV-Vis absorption spectra of the aqueous solutions of water-soluble nanoparticles corresponding to Example 1 and Application Example 2 were tested.

[0152] Test method: Weigh 1 mg of each of Examples 1 and 2, respectively, and dissolve them in ultrapure aqueous solution to prepare a concentration of 0.05 mg / mL. -1 The aqueous solution of nanoparticles was obtained, and the final aqueous solution of nanoparticles was analyzed by ultraviolet-visible-near-infrared absorption spectroscopy using a UV-Vis spectrophotometer.

[0153] The results are as follows Figure 3 As shown.

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

[0155] in,

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

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

[0158] The test results above show that the main absorption band of the water-soluble nanoparticle aqueous solution in Application Example 1 is in the range of 750-1320 nm, with a peak at 982 nm. The water-soluble nanoparticle aqueous solution in Application Example 2 has an absorption band centered at 975 nm in the near-infrared window. The absorption bands of the water-soluble nanoparticle aqueous solutions in both Application Example 1 and Application Example 2 can extend into the near-infrared II window.

[0159] Test Example 2

[0160] The stability of PCDT10V-SO-BBT from Example 1 and PCDT10V-SO-TBZ10V from Example 2 in aqueous solution and PBS solution was tested.

[0161] Test method: PCDT10V-SO-BBT from Example 1 and PCDT10V-SO-TBZ10V from Example 2 were dissolved in ultrapure water and PBS solution, respectively, to prepare test solutions with a concentration of 0.05 mg / mL. The particle size was measured every 3 days using a nanoparticle size analyzer for 15 days.

[0162] Test results are as follows Figure 4 As shown.

[0163] Figure 4 The stability of Examples 1 and 2 in aqueous solution and PBS is shown;

[0164] in,

[0165] Figure 4 (a) shows the stability of PCDT10V-SO-BBT for 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] The test results above show 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 clarity and color.

[0168] It is evident that the polymer of the present invention exhibits good solubility and stability in different media, and has the potential to serve as a near-infrared photoacoustic contrast agent.

[0169] Test Example 3

[0170] The size and zeta potential of the aqueous solution of water-soluble nanoparticles were tested in accordance with Use Case 1 and Application Example 2.

[0171] Test method: Weigh 1 mg of each of Examples 1 and 2, respectively, and dissolve them in ultrapure aqueous solution to prepare a concentration of 0.05 mg / mL. -1 The prepared aqueous solutions of nanoparticles were placed in separate cells, and the size and zeta potential of the aqueous solutions were measured using a Nano-ZS90.

[0172] Test results are as follows Figure 5 As shown.

[0173] Figure 5 Dynamic scattering patterns and Zeta potential diagrams of water-soluble nanoparticles in Application Examples 1 and 2 are shown.

[0174] in,

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

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

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

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

[0179] The test results above show that the water-soluble nanoparticles in Application Examples 1 and 2 have a particle size range of 150-250 nm, and their Zeta potentials are -12.1 mV and -34.9 mV, respectively. Experimental data indicate that Application Examples 1 and 2 possess excellent stability, which is beneficial for long-term in vivo blood circulation. This suggests that the water-soluble nanoparticles in Application Examples 1 and 2 have the potential to serve as photoacoustic contrast agents in the near-infrared region.

[0180] Test Example 4

[0181] Given that Application Example 1 and Application Example 2 have strong absorption in the near-infrared window region, the photothermal performance of Application Example 1 and Application Example 2 was tested through photothermal experiments.

[0182] Test method: The polymers from Examples 1 and 2 were weighed separately, added to ultrapure water, and dissolved thoroughly by sonication to prepare 200 μg / mL solutions. -1 and 400 μg mL -1 An aqueous solution of nanoparticles was added to a 600 μL centrifuge tube. The centrifuge tube and laser were fixed in place, and then a 1064 nm laser (2 W cm⁻¹) was used. -2 After irradiating the centrifuge tubes for 10 minutes, the laser was turned off and the solution was cooled to room temperature. The solution temperature was recorded every 30 seconds throughout the process. The temperature of the solution under 1064 nm laser light (1W cm⁻¹) was then calculated. -2 The photothermal conversion efficiency of CDT10V-SO-BBT and CDT10V-SO-TBZ under irradiation was calculated. Finally, the photothermal conversion efficiency (PCE) was calculated using formula (1):

[0183]

[0184] Where h and S are the heat transfer coefficient and the container surface area, respectively. Max and TSur These are the highest steady-state temperature and the ambient temperature, respectively. Q Dis This is the heat dissipated by the laser mediated by the solvent and 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 Given the mass of water (0.6 g) and its heat capacity (4.2 J / g), τ s The time constant for heat transfer in the system 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, which can be determined by formula (4):

[0189]

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

[0191] Test results are as follows Figure 6 As shown.

[0192] Figure 6 The temperature change curves of Application Example 1 and Application Example 2 with different concentrations of 1064nm laser irradiation are shown in the photothermal experiment.

[0193] in,

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

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

[0196] like Figure 6 As shown, in NIR-II laser (1064nm, 2W cm⁻¹) -2 After irradiation for 10 minutes, the temperature of the aqueous solutions in Application Examples 1 and 2 gradually increased, while the temperature of the ultrapure aqueous solution group did not show a significant change. Application Examples 1 and 2 were irradiated at 400 μg / mL... -1At these concentrations, temperatures reached 65.3°C and 64.8°C respectively, sufficient to cause irreversible thermal damage to the tumor. Furthermore, Application Examples 1 and 2 demonstrated this effect using a laser (1064nm, 2W cm⁻¹). -2 During the five cycles of the on / off process, the rising and cooling trends of the solution temperature did not change significantly. These data indicate that Application Example 1 and Application Example 2 exhibit excellent stability and high photothermal conversion efficiency for tumor photothermal therapy.

[0197] Test Example 5

[0198] Biocompatibility is crucial for PA imaging. Therefore, the biocompatibility of Application Examples 1 and 2 was assessed by in vitro cytotoxicity and hemolysis assays before they were used for in vivo PA imaging.

[0199] Test method:

[0200] (1) In vitro cytotoxicity assay: In the in vitro cytotoxicity assay, A549 and HepG2 cells (given to Gannan Medical University, the cells are existing technology) were treated with different concentrations of PCDT10V-SO-BBT@NPs for 24 h, and cell viability was assessed by MTT assay. Simultaneously, HepG2 and AML-12 cells (given to Gannan Medical University, the cells are existing technology) were treated with different concentrations of PCDT10V-SO-TBZ10V@NPs for 24 h, and cell viability was assessed by MTT assay.

[0201] (2) Hemolysis test: Application Example 1 and Application Example 2 with different concentrations were incubated with 4% red blood cell suspension for 1 hour and the hemolysis rate was measured.

[0202] Test results are as follows Figure 7 and Figure 8 As shown.

[0203] Figure 7 The cytotoxicity test results for Application Example 1 and Application Example 2 are shown.

[0204] in,

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

[0206] Figure 7 (b) shows a cytotoxicity test diagram of the nanoparticles PCDT10V-SO-TBZ10V@NPs in Application Example 2.

[0207] Figure 8 The hemolysis test diagrams for Application Example 1 and Application Example 2 are shown;

[0208] in,

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

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

[0211] Application Example 1: Results of in vitro cytotoxicity experiment as follows Figure 7 As shown in (a), after 24 h of incubation, at 0-160 μg / mL -1 At certain concentrations, cell viability gradually decreased with increasing PCDT10V-SO-BBT@NPs concentration. However, even at a concentration of 160 μg / mL... -1 The survival rate of HeLa cells remained above 90%. Example 2 shows the results of the in vitro cytotoxicity experiment. Figure 7 As shown in (b), at a concentration of 0-160 μg mL -1 Within a certain range, both cell types maintained good viability. Even at 160 μg / mL... -1 At the concentration of [specific concentration], the survival rate of Hep G2 cells and AML-12 cells still exceeded 90%.

[0212] The hemolytic activity of Application Examples 1 and 2 was further evaluated using a hemolysis test, a prerequisite for in vivo application. The results of the hemolysis test are as follows: Figure 8 As shown, both are even at a concentration of 160 μg / mL -1 No significant red blood cell lysis was observed, and the hemolysis rate remained less than 5%.

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

[0214] Test Example 6

[0215] Given 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 testing. A solid model (containing 2 wt% agar, 1 wt% endolipids, and the remainder water) was used to simulate in vitro conditions, and the PA signal intensity of water-soluble nanoparticles from Application Example 1 at different concentrations was measured.

[0216] Test method: In the in vitro experiment, a solid model containing agar (2 wt%), endolipids (1 wt%), and the balance water was first prepared. Then, 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 The solution is injected into a transparent quartz tube with a diameter of 0.9 mm, and then the two ends of the quartz tube are sealed and embedded in a solid model.

[0217] Subsequently, the solid model was placed on the experimental platform at an energy density of 20 mJ·cm⁻¹. -2 The PA image was obtained by detecting the image under laser irradiation using a PA imaging system. The scanning length was 10 mm, and the scanning steps were 100 μm.

[0218] Test results are as follows Figure 9 As shown.

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

[0220] in,

[0221] Figure 9 (a) shows that PCDT10V-SO-BBT@NPs can operate at a laser energy density of 20 mJ·cm⁻¹. -2 Photoacoustic signals at different concentrations under Nd:YAG laser irradiation (wavelength 1064nm);

[0222] Figure 9 (b) This demonstrates the performance of PCDT10V-SO-BBT@NPs at a laser energy density of 20 mJ·cm⁻¹. -2 The photoacoustic signal intensity at different concentrations under Nd:YAG laser irradiation (wavelength 1064nm).

[0223] from Figure 9 As can be seen in (a) and 9(b), as the concentration of the nanoparticle solution increases from 0.5 mg·mL⁻¹, the overall effect is different. -1 Rise to 2 mg / mL -1 The PA signal-to-noise ratio of PCDT10V-SO-BBT@NPs increased. Furthermore, an almost positive correlation was observed between the imaging signal and the concentration of PCDT10V-SO-BBT@NPs. And at a concentration of 2 mg / mL... -1 It exhibits the strongest PA signal-to-noise ratio and has the capability for in vivo PA imaging.

[0224] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0225] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider 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 DA-type conjugated polymer based on click chemistry, characterized in that, The click chemistry-based DA-type conjugated polymer has the following structure: ； in, The n is selected from positive integers from 1 to 10000.

2. The method for preparing the DA-type conjugated polymer based on click chemistry as described in claim 1, characterized in that, Includes the following steps: S1. Under the protection of an inert gas, cyclopentylthiophene and haloalkene were blended, stirred, and purified to obtain intermediate product 1. S2. Under the protection of an inert gas, the intermediate product 1, the organolithium compound and the organotin compound are mixed and reacted, and then purified to obtain intermediate product 2. S3. Under the protection of an inert gas, the intermediate product 2 and the monomer containing an electron-withdrawing unit are blended, a catalyst is added, the reaction is heated, and the mixture is purified to obtain the intermediate product 3. S4. Under the protection of an inert gas, the intermediate product 3 and the thiol hydrophilic compound are blended, reacted, and purified to obtain a DA-type conjugated polymer based on click chemistry.

3. The method for preparing DA-type conjugated polymers based on click chemistry according to claim 2, characterized in that, In step S3, the heating temperature is 130-150℃.

4. The method for preparing DA-type conjugated polymers based on click chemistry according to claim 2, characterized in that, The method for preparing the monomer containing the electron-withdrawing unit includes the following steps: L1. Under the protection of an inert gas, benzothiadiazole or its derivative containing a nitro group is blended with an aliphatic carboxylic acid, heated, a reducing agent is added, the reaction is carried out, and the mixture is purified to obtain intermediate 1. L2. Under the protection of an inert gas, intermediate 1 and nitrite are mixed, stirred and reacted, and purified to obtain intermediate 2. L3. Under the protection of an inert gas, intermediate 2, haloolefin and tert-butyl alkoxide are blended, heated and reacted, and purified to obtain a monomer containing an electron-withdrawing unit.

5. A water-soluble nanoparticle, characterized in that, The water-soluble nanoparticles comprise the DA-type conjugated polymer based on click chemistry as described in claim 1, and are formed by dissolving it in a solvent.

6. The water-soluble nanoparticles according to claim 5, characterized in that, The water-soluble nanoparticles have a particle size of 150-250 nm.

7. The water-soluble nanoparticles according to claim 5, characterized in that, The mass ratio of the click chemistry-based DA-type conjugated polymer to the solvent is 1:(1-2).

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