Tumor-targeting water-soluble conjugated polymer contrast agent, preparation method and application

Abstract

The present invention discloses a tumor-targeting water-soluble conjugated polymer contrast agent, a preparation method, and an application. The contrast agent has a chemical structure shown in the following formula III: #imgabs0# wherein ‑NH‑R is an amino acid side chain obtained by removing one hydrogen from an amino acid. The side chains of the tumor-targeting water-soluble conjugated polymer contrast agent provided by the present invention are selected from three branched-chain amino acids commonly found in proteins to provide nutrients required for abnormal tumor proliferation. The polyisoleucine, polyvaline, and polyleucine contained in the side ends of the polymer are covalently linked to each other, which not only provide a large number of amino groups, making the near-infrared second-region conjugated polymer have good water solubility, but more importantly, can achieve tumor targeting, thereby reducing cytotoxicity, enhancing cellular uptake of the material, and increasing the enrichment rate of the material, thereby improving the effect of fluorescence imaging.

Description

Technical Field

[0001] The present invention relates to the technical field of nano-biomedical photosensitivity diagnosis and treatment, and in particular to a tumor-targeting water-soluble conjugated polymer contrast agent, a preparation method and applications thereof. Background Art

[0002] Malignant tumors, also known as cancer, are one of the greatest threats facing humanity today. Along with heart disease and cerebrovascular disease, they are among the top three causes of death. Malignant tumors, due to their diverse types, complexity, and hidden early symptoms, lead to a high mortality rate. Therefore, early diagnosis of cancer has become a hot research topic.

[0003] In recent years, various imaging techniques, including computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT), have been used for in vivo imaging. However, due to insufficient spatial and temporal resolution and safety risks, it is difficult to achieve simultaneous in situ, real-time imaging of multiple events in vivo. Fluorescence imaging in the second near-infrared window (NIR-II, 1000-1700 nm) is an emerging technology that has deeper tissue penetration, higher spatial resolution, and higher temporal resolution compared to traditional fluorescence imaging (400-900 nm) because it reduces photon absorption and scattering and can ignore tissue autofluorescence.

[0004] Traditional targeting strategies include passive and active targeting. Passive targeting involves adjusting the size and surface chemistry of nanoparticles to enhance permeability and retention (EPR) effects, promoting their selective accumulation in tumors. However, its drawbacks include limited enrichment and high particle size requirements. Active targeting involves binding high-affinity ligands, such as peptides, proteins, and aptamers, to specific surface molecules primarily expressed by cancer cells or tumor epithelial cells. However, the cost of peptides and proteins is relatively high. Improving the efficiency of enrichment lies in enhancing the specific targeting of contrast agents. Traditional contrast agents are encapsulated in polyethylene glycol (PEG) for cell entry and tumor targeting via the EPR effect. PEG, with its neutral, water-soluble groups, lacks specific recognition for other molecules or structures, resulting in limited enrichment. Furthermore, only a few organic molecules have been shown to emit fluorescence in the NIR-II region. These are highly hydrophobic, water-insoluble dyes that must be encapsulated in a polymer matrix for bioimaging, requiring their particle size to exceed the renal filtration threshold (ca. 40 kDa). Recently, the small molecule CH1055 (8.9 kDa) NIR-II organic dye, which emits at approximately 1055 nm under 808 nm excitation and has high water solubility, has shown promise in preclinical applications. However, research on NIR-II organic dyes with simple preparation processes, high quantum yields, and high-resolution imaging quality is still in its infancy. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a tumor-targeting water-soluble conjugated polymer contrast agent, a preparation method and an application thereof, in view of the deficiencies in the above-mentioned prior art.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows: In a first aspect of the present invention, a tumor-targeting water-soluble conjugated polymer contrast agent is provided, which has a chemical structure shown in the following formula III:

[0007]

[0008] Wherein, -NH-R is an amino acid side chain obtained by removing one hydrogen from the amino group in the amino acid, x=y=4-8, and m=30-50.

[0009] The targeted water-soluble conjugated polymer contrast agent of the present invention has an amphiphilic molecular structure, containing a polymer main chain and a side chain, wherein the polymer main chain has a donor and an acceptor structure, which can extend the conjugated bond length in the conjugated skeleton, red-shift the absorption, increase the fluorescence intensity of the polymer, and enhance the resolution and contrast of in vivo imaging; the side chain selects three common branched-chain amino acids in proteins to provide nutrients required for abnormal tumor proliferation, and is covalently linked to the side end of the polymer. The polyisoleucine, polyvaline, and polyleucine contained therein can not only provide a large number of amino groups, so that the near-infrared second region conjugated polymer has good water solubility, but more importantly, it can achieve tumor targeting, thereby reducing cytotoxicity, enhancing cellular uptake of the material, and increasing the enrichment rate of the material, thereby improving the effect of fluorescence imaging. The above-mentioned characteristics of the polymer enable it to have advantages such as improved lesion targeting enrichment, signal-to-noise ratio, and imaging clarity when used for fluorescence imaging.

[0010] The structure shown in the above formula I and the end-blocking group connected to the thiophene ring are The end-capping group connected to the benzene ring is a halogenated group, such as a bromo group.

[0011] A second aspect of the present invention provides a method for preparing the above-mentioned tumor-targeting water-soluble conjugated polymer contrast agent, comprising the following steps:

[0012] S1, prepare polymer 1 having the structure shown in the following formula I:

[0013]

[0014] Where, x=y=4-8;

[0015] S2. Atom transfer radical polymerization is performed on polymer 1 and N-succinimidyl acrylate to prepare polymer 2 having a structure shown in the following formula II:

[0016]

[0017] Wherein, m = 30 to 50;

[0018] S3. Then, the succinimide ester of polymer 2 is removed and amidation reaction is carried out with the amino group of amino acid to prepare polymer 3, i.e., the tumor-targeting water-soluble conjugated polymer contrast agent, whose structural formula is shown in Formula III.

[0019] Wherein, the amino acid is any one of isoleucine, valine and leucine.

[0020] The targeted water-soluble conjugated polymer contrast agent of the present invention has an amphiphilic molecular structure, containing a polymer main chain and a side chain, wherein the polymer main chain has a donor and an acceptor structure, which can extend the conjugated bond length in the conjugated skeleton, red-shift the absorption, increase the fluorescence intensity of the polymer, and enhance the resolution and contrast of in vivo imaging; the side chain selects three common branched-chain amino acids in proteins to provide nutrients required for abnormal tumor proliferation, and is covalently linked to the side end of the polymer. The polyisoleucine, polyvaline, and polyleucine contained therein can not only provide a large number of amino groups, so that the near-infrared second region conjugated polymer has good water solubility, but more importantly, it can achieve tumor targeting, thereby reducing cytotoxicity, enhancing cellular uptake of the material, and increasing the enrichment rate of the material, thereby improving the effect of fluorescence imaging. The above-mentioned characteristics of the polymer enable it to have advantages such as improved lesion targeting enrichment, signal-to-noise ratio, and imaging clarity when used for fluorescence imaging.

[0021] For example, in the structure shown in Formula II, the end-capping group connected to the thiophene ring is The end-capping group connected to the benzene ring is a halide group, for example, a bromo group.

[0022] Preferably, in step S1, the polymer 1 is prepared by subjecting a monomer 1 having a structure as shown in formula IV, a monomer 2 having a structure as shown in formula V, and a monomer 3 having a structure as shown in formula VI to a Stiller coupling reaction. The synthesis route is as follows:

[0023]

[0024] Specifically, monomer 1 is: 4,7-dibromobenzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole, with the structural formula IV;

[0025] Monomer 2 is: 2,6-bis(trimethyltin)-4,8-bis(5-(2-ethylhexyl)thiophen-2-yl)-benzodithiophene, with the structural formula V;

[0026] Monomer 3 is: (2,7-dibromo-9H-fluorene-9,9-diyl)bis(propane-3,1-diyl)bis(2-bromo-2-methylpropionate), and has the structural formula of Formula VI.

[0027] Preferably, step S1 is specifically as follows: in a light-proof environment, monomer 1, monomer 2, and monomer 3 are added to a solvent and completely dissolved, and then nitrogen is bubbled into the resulting solution for more than 20 minutes, a palladium catalyst is added to the solution, and the mixture is reacted under nitrogen protection at 80-90° C. for 2-4 hours. After the reaction is completed, the product is filtered and then precipitated in methanol to obtain the polymer 1.

[0028] Preferably, in step S1, the molar ratio of monomer 1:monomer 2:monomer 3 is 1:2:1.

[0029] Preferably, step S1 is specifically as follows: in a light-proof environment, 0.2 mM monomer 1, 0.2 mM monomer 2, and 0.1 mM monomer 3 are added to 1 mL of toluene and completely dissolved, and then nitrogen is bubbled into the resulting solution for more than 20 minutes, 50 mg of bis(triphenylphosphine)palladium(II) dichloride is added to the solution, and the mixture is reacted at 80° C. under nitrogen protection for 3 hours. After the reaction, the product is filtered and then repeatedly precipitated in methanol to obtain the polymer 1. The palladium catalyst is preferably bis(triphenylphosphine)palladium(II) dichloride, and the solvent is preferably toluene.

[0030] Preferably, the step S2 is specifically as follows:

[0031] Under light-shielding conditions, polymer 1 and acrylic acid-N-succinimidyl ester were added to an organic solvent, followed by a catalyst, and pentamethyldiethylenetriamine was added under nitrogen protection. The resulting mixed solution was reacted at 80-100° C. for 9-12 hours. After the reaction, the product was filtered and then repeatedly precipitated in diethyl ether to obtain polymer 2. The synthesis route is as follows:

[0032]

[0033] Preferably, the molar ratio of polymer 1 to N-succinimidyl acrylate is 1:50 to 200, for example, 1:100.

[0034] Preferably, the catalyst is cuprous bromide and the organic solvent is anisole.

[0035] Preferably, the step S2 is specifically as follows:

[0036] Under light-shielding conditions, 100 mg of polymer 1 and 887 mg of acrylic acid-N-succinimidyl ester were added to 1 mL of anisole, and then the catalyst CuBr was added. Under nitrogen protection, 50 μL of pentamethyldiethylenetriamine was added, and the resulting mixed solution was reacted at 90° C. for 12 h. After the reaction, the product was filtered and then repeatedly precipitated in diethyl ether to obtain the polymer 2.

[0037] Preferably, the step S3 is specifically as follows:

[0038] Under light-shielding conditions, polymer 2 and amino acid were dissolved in DMF, triethylamine was added under nitrogen protection, and the reaction was stirred at 35-45°C for 24-60 hours. After the reaction, the solution in the product system was added to deionized water, and the resulting mixed aqueous solution was placed in a dialysis bag and dialyzed with deionized water. After the dialysis, the solution in the dialysis bag was dried to obtain polymer 3.

[0039] Preferably, the step S3 is specifically as follows:

[0040] Under light-shielding conditions, 300 mg of polymer 2 and 1.75 mmol of isoleucine were dissolved in 5 mL of DMF. Under nitrogen protection, 1.75 mmol of triethylamine was added, and the mixture was stirred at 40°C for 48 hours. After the reaction, the solution in the product system was added to 20 mL of deionized water. The resulting mixed aqueous solution was placed in a dialysis bag with a molecular weight cutoff of 3500 and dialyzed with deionized water for 48 hours, with the dialysate being changed every 6 hours. After the dialysis, the solution in the dialysis bag was freeze-dried to obtain polymer 3 having the chemical structure shown in Formula III-1. The synthetic route is:

[0041]

[0042] Preferably, the step S3 is specifically as follows:

[0043] Under light-shielding conditions, 300 mg of polymer 2 and 1.75 mmol of valine were dissolved in 5 mL of DMF. Under nitrogen protection, 1.75 mmol of triethylamine was added, and the mixture was stirred at 40°C for 48 hours. After the reaction, the solution in the product system was added to 20 mL of deionized water. The resulting mixed aqueous solution was placed in a dialysis bag with a molecular weight cutoff of 3500 and dialyzed with deionized water for 48 hours. The dialyzate was changed every 6 hours. After the dialysis, the solution in the dialysis bag was freeze-dried to obtain polymer 3 having the chemical structure shown in Formula III-2. The synthetic route is:

[0044]

[0045] Preferably, the step S3 is specifically as follows:

[0046] Under light-shielding conditions, 300 mg of polymer 2 and 1.75 mmol of leucine were dissolved in 5 mL of DMF. Under nitrogen protection, 1.75 mmol of triethylamine was added, and the mixture was stirred at 40°C for 48 hours. After the reaction, the solution in the product system was added to 20 mL of deionized water. The resulting mixed aqueous solution was placed in a dialysis bag with a molecular weight cutoff of 3500 and dialyzed with deionized water for 48 hours. The dialyzate was changed every 6 hours. After the dialysis, the solution in the dialysis bag was freeze-dried to obtain polymer 3 having the chemical structure shown in Formula III-3. The synthetic route is:

[0047]

[0048] The third aspect of the present invention provides a use of the above-mentioned tumor-targeting water-soluble conjugated polymer contrast agent in near-infrared second window fluorescence imaging.

[0049] The beneficial effects of the present invention are:

[0050] The tumor-targeting water-soluble conjugated polymer contrast agent provided by the present invention has an amphiphilic molecular structure, containing a polymer backbone and a side chain, wherein the polymer backbone has a donor and an acceptor structure, which can extend the conjugated bond length in the conjugated skeleton, red-shift the absorption, improve the fluorescence intensity of the polymer, and enhance the resolution and contrast of in vivo imaging; the side chain selects three common branched-chain amino acids in proteins to provide the nutrients required for abnormal tumor proliferation, and is covalently linked to the polymer side end, containing polyisoleucine, polyvaline and polyleucine, which can not only provide a large number of amino groups, but also make the near-infrared second-zone conjugated polymer have good water solubility. More importantly, it can achieve tumor targeting, thereby reducing cytotoxicity, enhancing cellular uptake of materials, and increasing the enrichment rate of materials, thereby improving the effect of fluorescence imaging. The above-mentioned characteristics of the polymer enable it to have the advantages of improving lesion targeting enrichment, signal-to-noise ratio and imaging clarity when used for fluorescence imaging, so that it can be well applied to near-infrared second window fluorescence imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 is the absorption spectrum of polymer 1 prepared in Example 1;

[0052] Figure 2 is the absorption spectrum of polymer 2 prepared in Example 1;

[0053] Figure 3 1H NMR spectra of polymer 3-1, polymer 3-2, and polymer 3-3 prepared in Examples 2 to 4;

[0054] Figure 4 Transmission electron micrographs of polymer 3-1, polymer 3-2, and polymer 3-3 prepared in Examples 2 to 4;

[0055] Figure 5 Schematic diagram of the nanometer diameters of polymers 3-1, 3-2, and 3-3 prepared in Examples 2 to 4 measured by dynamic light scattering;

[0056] Figure 6 The absorption and emission spectra (1 mg / mL) of polymer 3-1, polymer 3-2, and polymer 3-3 prepared in Examples 2 to 4 are shown;

[0057] Figure 7 These are cell confocal imaging images of polymers 3-1, 3-2, and 3-3 prepared in Examples 2 to 4;

[0058] Figure 8 Quantitative analysis of fluorescence intensity of cell confocal imaging images of polymers 3-1, 3-2, and 3-3 prepared in Examples 2 to 4;

[0059] Figure 9 This is a second window near-infrared fluorescence imaging image of the targeted water-soluble conjugated polymer contrast agent aqueous solution prepared in Example 2;

[0060] Figure 10 This is a second window near-infrared fluorescence imaging image of the targeted water-soluble conjugated polymer contrast agent aqueous solution prepared in Example 3;

[0061] Figure 11 This is a second window near-infrared fluorescence imaging image of the targeted water-soluble conjugated polymer contrast agent aqueous solution prepared in Example 4;

[0062] Figure 12 This is a cell survival rate graph of the cytotoxicity of the targeted water-soluble conjugated polymer prepared in Example 2 on mouse breast cancer cells;

[0063] Figure 13 This is a cell survival rate graph of the cytotoxicity of the targeted water-soluble conjugated polymer prepared in Example 3 to mouse breast cancer cells;

[0064] Figure 14 This is a cell survival chart showing the cytotoxicity of the targeted water-soluble conjugated polymer prepared in Example 4 to mouse breast cancer cells. DETAILED DESCRIPTION

[0065] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.

[0066] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0067] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Materials and reagents used in the following examples are commercially available unless otherwise specified. In the following examples, where specific conditions are not specified, the experiments were conducted under conventional conditions or those recommended by the manufacturer. Reagents and instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0068] Example 1 Preparation of Polymer 1 and Polymer 2

[0069] 1. The chemical structure of polymer 1 is as follows:

[0070]

[0071] Where, x=y=4-8;

[0072] The preparation method is:

[0073] Under light-proof environment, the acceptor molecule (monomer 1) 4,7-dibromobenzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole (35.2 mg, 0.2 mM), the donor molecule (monomer 2) 2,6-bis(trimethyltin)-4,8-bis(5-(2-ethylhexyl)thiophen-2-yl)-benzodithiophene (180.9 mg, 0.2 mM), and the donor molecule (monomer 3) (2,7-dibromo-9H-fluorene-9,9-diyl)bis( ...6-ditrimethyltin)-4,8-bis(2,6-ditrimethyltin)-4,8-bis(2,6-ditrimethyltin)- (Propan-3,1-diyl)bis(2-bromo-2-methylpropionate) (73.8 mg, 0.1 mM) was dissolved in toluene (1 mL) and nitrogen was bubbled in for more than 20 minutes; bis(triphenylphosphine)palladium(II) dichloride (50 mg) was quickly added to the flask, and the flask was placed at 80°C under nitrogen protection to react for 3 hours; the crude product was filtered with an organic filter membrane to remove the solid catalyst, and then repeatedly precipitated in methanol to obtain a reddish-brown polymer 1 (100 mg).

[0074] 2. The chemical structure of polymer 2 is as follows:

[0075]

[0076] Wherein, m = 30 to 50;

[0077] The preparation method is:

[0078] Under light-shielding conditions, polymer 1 (100 mg) and acrylic acid-N-succinimide ester (887 mg, 5.24 mM) were added to a reaction tube, the above solid was dissolved with anisole (1 mL), CuBr was added as a catalyst, and under nitrogen protection, 50 μL of pentamethyldiethylenetriamine was added as a ligand. The resulting mixed solution was reacted at 90° C. for 12 h. After the reaction, it was filtered with an organic filter membrane to remove the solid catalyst, and then repeatedly precipitated in ether to obtain a reddish-brown conjugated main chain polymer substituted with acrylic acid-N-succinimide ester side chains, polymer 2 (900 mg).

[0079] Polymer 1 and Polymer 2 in the following Examples 2-4 were prepared by this example.

[0080] Example 2

[0081] A tumor-targeting water-soluble conjugated polymer contrast agent, the chemical structure of which is shown in the following formula III-1:

[0082]

[0083] Among them, x=y=4~8, m=30~50.

[0084] The -NH-R is an amino acid side chain obtained by removing a hydrogen from the amino group of isoleucine.

[0085] The preparation method is as follows: 300 mg of polymer 2 and 1.75 mmol of valine are dissolved in 5 mL of DMF under light-shielding conditions, 1.75 mmol of triethylamine is added under nitrogen protection, and the mixture is stirred at 40° C. for 48 hours. After the reaction, the solution in the product system is added to 20 mL of deionized water, and the resulting mixed aqueous solution is placed in a dialysis bag with a molecular weight cutoff of 3500 and dialyzed with deionized water for 48 hours, with the dialysate being changed every 6 hours. After the dialysis, the solution in the dialysis bag is freeze-dried to obtain a polymer having a chemical structure shown in Formula III-2, which is recorded as polymer 3-1. The synthetic route is:

[0086]

[0087] Example 3

[0088] A tumor-targeting water-soluble conjugated polymer contrast agent, the chemical structure of which is shown in Formula III-2 below:

[0089] Among them, x=y=4~8, m=30~50.

[0090] The -NH-R is an amino acid side chain obtained by removing a hydrogen from the amino group of valine.

[0091] Its preparation method is:

[0092] Under light-shielding conditions, 300 mg of polymer 2 and 1.75 mmol of valine were dissolved in 5 mL of DMF. Under nitrogen protection, 1.75 mmol of triethylamine was added, and the reaction was stirred at 40°C for 48 hours. After the reaction, the solution in the product system was added to 20 mL of deionized water. The resulting mixed aqueous solution was placed in a dialysis bag with a molecular weight cutoff of 3500 and dialyzed with deionized water for 48 hours. The dialysate was changed every 6 hours. After the dialysis, the solution in the dialysis bag was freeze-dried to obtain a polymer having the chemical structure shown in Formula III-2, which was recorded as polymer 3-2. The synthetic route is:

[0093]

[0094] Example 4

[0095] A tumor-targeting water-soluble conjugated polymer contrast agent, the chemical structure of which is shown in Formula III-3 below:

[0096]

[0097] Among them, x=y=4~8, m=30~50.

[0098] The -NH-R is an amino acid side chain obtained by removing a hydrogen from the amino group of leucine.

[0099] Its preparation method is:

[0100] Under light-shielding conditions, 300 mg of polymer 2 and 1.75 mmol of leucine were dissolved in 5 mL of DMF. Under nitrogen protection, 1.75 mmol of triethylamine was added, and the reaction was stirred at 40°C for 48 hours. After the reaction, the solution in the product system was added to 20 mL of deionized water. The resulting mixed aqueous solution was placed in a dialysis bag with a molecular weight cutoff of 3500 and dialyzed with deionized water for 48 hours. The dialysate was changed every 6 hours. After the dialysis, the solution in the dialysis bag was freeze-dried to obtain a polymer having the chemical structure shown in Formula III-3, which was recorded as polymer 3-3. The synthesis route is:

[0101]

[0102] The products prepared in Examples 1-4 were subjected to the following performance tests and characterizations:

[0103] 1. Using dichloromethane as an organic solvent, prepare an organic solution of polymer 1 prepared in Example 1 and test its absorption spectrum. Figure 1 As shown, it can be seen that its absorption peak is located in the 734 nm band.

[0104] 2. Using dichloromethane as an organic solvent, prepare an organic solution of polymer 2 prepared in Example 1 and test its absorption spectrum. Figure 2 As shown, it can be seen that its absorption peak is located in the 727 nm band.

[0105] 3. The tumor-targeting water-soluble conjugated polymer contrast agents prepared in Examples 2 to 4 (respectively denoted as polymer 3-1, polymer 3-2, and polymer 3-3) were subjected to nuclear magnetic resonance spectroscopy to obtain the nuclear magnetic resonance hydrogen spectrum of the targeted water-soluble conjugated polymer contrast agent, as shown in FIG. Figure 3 As shown, characteristic proton signals of amino acid side chains can be seen.

[0106] 4. The tumor-targeting water-soluble conjugated polymer contrast agents prepared in Examples 2 to 4 (referred to as polymer 3-1, polymer 3-2, and polymer 3-3, respectively) were prepared into aqueous polymer solutions with a concentration of 1 mg / mL, and the following tests were then performed:

[0107] i) Each aqueous solution was analyzed using a transmission electron microscope, and the resulting transmission electron microscope images are shown in FIG. Figure 4 As shown by Figure 4 It can be seen that the particle sizes of polymer 3-1, polymer 3-2, and polymer 3-3 are all about 100 nanometers.

[0108] ii) Based on the prepared polymer aqueous solutions, dynamic light scattering measurements showed that the average hydrodynamic diameter of polymer 3-1 in water was approximately 102 nm, the average hydrodynamic diameter of polymer 3-2 in water was approximately 90 nm, and the average hydrodynamic diameter of polymer 3-3 in water was approximately 90 nm. The obtained hydrodynamic volume diagrams of each molecule are shown in FIG. Figure 5 shown.

[0109] iii) Based on the polymer aqueous solution, the absorption and emission spectra of polymer 3-1, polymer 3-2 and polymer 3-3 were tested respectively. The results are as follows Figure 6 As shown, it can be seen that: the absorption peak of polymer 3-1 is located in the 970 nm band, and its emission peak is located in the 1047 nm band under 808 nm laser excitation; the absorption peak of polymer 3-2 is located in the 958 nm band, and its emission peak is located in the 1048 nm band under 808 nm laser excitation; the absorption peak of polymer 3-3 is located in the 926 nm band, and its emission peak is located in the 1069 nm band under 808 nm laser excitation.

[0110] 5. The polymers 3-1, 3-2 and 3-3 prepared in Examples 2 to 4 were respectively prepared into aqueous solutions with concentration gradients of 2 mg / mL, 1 mg / mL, 0.5 mg / mL and 0.25 mg / mL. The solutions were excited with a 1064 nm laser and fluorescence images were taken in a second near-infrared window fluorescence imager. The results are shown in FIG. Figures 9-11 As shown, polymer 3-1, polymer 3-2, and polymer 3-3 all have the performance of near-infrared second-zone fluorescence imaging.

[0111] 6. Dissolve 10 mg of each freeze-dried solid powder of polymer 3-1, polymer 3-2, and polymer 3-3 prepared in Examples 2 to 4 in a small amount of dimethyl sulfoxide solution. Use nanoprecipitation to encapsulate Nile red dye. Detect the absorbance of Nile red at 552 nm to keep it at the same absorbance. Observe the fluorescence effect of the dye using a confocal microscope. Calculate the fluorescence intensity and compare the cellular uptake rate of different materials. The confocal microscope images are as follows: Figure 7 As shown in the figure, from top to bottom are the fluorescence image of the cell nucleus fluorescent dye, the fluorescence image of Nile red, and the superimposed fluorescence image; from left to right are polymer 3-1, polymer 3-2, and polymer 3-3. It can be seen that the fluorescence intensity of polymer 3-1, polymer 3-2, and polymer 3-3 is large, indicating that the targeted water-soluble conjugated polymer contrast agent prepared by the present invention has a large lesion enrichment and good cell targeting. Quantitative mean fluorescence intensity analysis is shown in Figure 2. Figure 8 As shown, it can be seen that the cell uptake rate of polymer 3-3 is the highest.

[0112] 7. Polymer 3-1, polymer 3-2, and polymer 3-3 prepared in Examples 2 to 4 were respectively formulated into a series of cell culture fluids with a certain concentration gradient, wherein the highest concentration of the polymer in the cell culture fluid was 2 mg / mL; mouse breast cancer cells were cultured with the above three culture fluids for 4 hours, CCK-8 reagent was added, and the absorbance at 450 nm was measured. The obtained absorbance was converted into cell survival rate, as shown in FIG. Figures 12-14 As shown, it can be seen that the cytotoxicity of polymer 3-1, polymer 3-2 and polymer 3-3 is very low, indicating that the targeted water-soluble conjugated polymer contrast agent prepared by the present invention has good biocompatibility.

[0113] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A tumor-targeting water-soluble conjugated polymer contrast agent, characterized in that: It has the chemical structure shown in the following formula III: ， Among them, -NH-R is the amino acid side chain obtained by removing one hydrogen from the amino acid, x=y=4~8, m=30~50.

2. A method for preparing the tumor-targeting water-soluble conjugated polymer contrast agent according to claim 1, characterized in that: The following steps are involved: S1, prepare polymer 1 having the structure shown in the following formula I: ， Where x = y = 4~8; S2. Atom transfer radical polymerization is performed on polymer 1 and N-succinimidyl acrylate to prepare polymer 2 having a structure shown in the following formula II: ， Among them, m=30~50; S3, then removing the succinimide ester from polymer 2 and subjecting it to an amidation reaction with the amino group of an amino acid to prepare polymer 3, i.e., the tumor-targeting water-soluble conjugated polymer contrast agent, whose structural formula is shown in Formula III; Wherein, the amino acid is any one of isoleucine, valine and leucine.

3. The method for preparing the tumor-targeting water-soluble conjugated polymer contrast agent according to claim 2, characterized in that: In step S1, the polymer 1 is prepared by subjecting a monomer 1 having a structure as shown in formula IV, a monomer 2 having a structure as shown in formula V, and a monomer 3 having a structure as shown in formula VI to a Stiller coupling reaction. The synthesis route is as follows: 。 4. The method for preparing the tumor-targeting water-soluble conjugated polymer contrast agent according to claim 3, characterized in that: The step S1 specifically comprises: adding monomer 1, monomer 2, and monomer 3 to a solvent and completely dissolving them in a light-proof environment, then bubbling nitrogen into the resulting solution for more than 20 minutes, adding a palladium catalyst to the solution, reacting at 80-90° C. under nitrogen protection for 2-4 hours, filtering the product after the reaction, and then settling it in methanol to obtain the polymer 1.

5. The method for preparing the tumor-targeting water-soluble conjugated polymer contrast agent according to claim 4, characterized in that: In step S1, the molar ratio of monomer 1:monomer 2:monomer 3 is 2:2:

1.

6. The method for preparing the tumor-targeting water-soluble conjugated polymer contrast agent according to claim 5, characterized in that: The step S1 specifically comprises: in a light-proof environment, adding 0.2 mM monomer 1, 0.2 mM monomer 2, and 0.1 mM monomer 3 to 1 mL of toluene and completely dissolving them, then bubbling nitrogen into the resulting solution for more than 20 minutes, adding 50 mg of bis(triphenylphosphine)palladium(II) dichloride to the solution, reacting at 80° C. under nitrogen protection for 3 hours, filtering the product after the reaction, and then repeatedly precipitating it in methanol to obtain the polymer 1.

7. The method for preparing the tumor-targeting water-soluble conjugated polymer contrast agent according to claim 3, characterized in that: The step S2 is specifically as follows: Under light-shielding conditions, polymer 1 and acrylic acid-N-succinimidyl ester were added to an organic solvent, followed by a catalyst. Pentamethyldiethylenetriamine was added under nitrogen protection, and the resulting mixed solution was reacted at 80-100°C for 9-12 hours. After the reaction, the product was filtered and then repeatedly precipitated in diethyl ether to obtain polymer 2. The synthetic route is as follows: 。 8. The method for preparing the tumor-targeting water-soluble conjugated polymer contrast agent according to claim 7, characterized in that: The step S2 is specifically as follows: Under light-shielding conditions, 100 mg of polymer 1 and 887 mg of acrylic acid-N-succinimidyl ester were added to 1 mL of anisole, and then the catalyst CuBr was added. Under nitrogen protection, 50 μL of pentamethyldiethylenetriamine was added, and the resulting mixed solution was reacted at 90°C for 12 h. After the reaction, the product was filtered and then repeatedly precipitated in diethyl ether to obtain the polymer 2.

9. The method for preparing the tumor-targeting water-soluble conjugated polymer contrast agent according to claim 2, characterized in that: The step S3 is specifically as follows: Under light-shielding conditions, polymer 2 and amino acid were dissolved in DMF. Under nitrogen protection, triethylamine was added, and the reaction was stirred at 35-45°C for 24-60 hours. After the reaction, the solution in the product system was added to deionized water, and the resulting mixed aqueous solution was placed in a dialysis bag and dialyzed with deionized water. After the dialysis, the solution in the dialysis bag was dried to obtain polymer 3.

10. The method for preparing the tumor-targeting water-soluble conjugated polymer contrast agent according to claim 9, characterized in that: The step S3 is specifically as follows: Under light-shielding conditions, 300 mg of polymer 2 and 1.75 mmol of isoleucine were dissolved in 5 mL of DMF. Under nitrogen protection, 1.75 mmol of triethylamine was added and the mixture was stirred at 40°C for 48 h. After the reaction, the solution in the product system was added to 20 mL of deionized water. The resulting mixed aqueous solution was placed in a dialysis bag with a molecular weight cutoff of 3500 and dialyzed against deionized water for 48 h. The dialyzate was changed every 6 h. After the dialysis, the solution in the dialysis bag was freeze-dried to obtain polymer 3 having the chemical structure shown in Formula III-1. The synthetic route is: 。 11. The method for preparing the tumor-targeting water-soluble conjugated polymer contrast agent according to claim 9, characterized in that: The step S3 is specifically as follows: Under light-shielding conditions, 300 mg of polymer 2 and 1.75 mmol of valine were dissolved in 5 mL of DMF. Under nitrogen protection, 1.75 mmol of triethylamine was added and the mixture was stirred at 40°C for 48 h. After the reaction, the solution in the product system was added to 20 mL of deionized water. The resulting mixed aqueous solution was placed in a dialysis bag with a molecular weight cutoff of 3500 and dialyzed with deionized water for 48 h. The dialyzate was changed every 6 h. After the dialysis, the solution in the dialysis bag was freeze-dried to obtain polymer 3 having the chemical structure shown in Formula III-2. The synthetic route is: 。 12. The method for preparing the tumor-targeting water-soluble conjugated polymer contrast agent according to claim 9, characterized in that: The step S3 is specifically as follows: Under light-shielding conditions, 300 mg of polymer 2 and 1.75 mmol of leucine were dissolved in 5 mL of DMF. Under nitrogen protection, 1.75 mmol of triethylamine was added, and the mixture was stirred at 40°C for 48 h. After the reaction, the solution in the product system was added to 20 mL of deionized water. The resulting mixed aqueous solution was placed in a dialysis bag with a molecular weight cutoff of 3500 and dialyzed with deionized water for 48 h. The dialyzate was changed every 6 h. After the dialysis, the solution in the dialysis bag was freeze-dried to obtain polymer 3 having the chemical structure shown in Formula III-3. The synthetic route is: 。 13. Use of the tumor-targeting water-soluble conjugated polymer contrast agent according to claim 1 or the tumor-targeting water-soluble conjugated polymer contrast agent prepared by the method according to any one of claims 2 to 12 in near-infrared second window fluorescence imaging.

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