Cyanine near-infrared contrast agent as well as preparation method and application thereof

Abstract

The invention relates to the field of nano biomedical photosensitive diagnosis and treatment, in particular to a cyanine near-infrared contrast agent as well as a preparation method and application thereof. The cyanine near-infrared contrast agent provided by the invention has an amphiphilic molecular structure, so that the stability of fluorescence can be ensured, and non-specific adsorption is reduced through a hydrophilic chain. Meanwhile, hyaluronic acid is specifically combined with a CD44 receptor on the surface of a tumor cell, so that a contrast agent can be guided to preferentially gather in a tumor tissue, the targeting property to the tumor tissue is improved, and the toxicity caused by dissociation of near-infrared cyanine dyes is reduced. In addition, interference of background fluorescence is reduced due to high tumor targeting, and the signal-to-noise ratio and the imaging effect of NIR-II are improved. Besides, hyaluronic acid has good water solubility, and after water-soluble modification is carried out on the cyanine molecules, the cyanine molecules have good biocompatibility, immune system recognition is reduced, and attack by an antibody or complement system is avoided.

Description

Technical Field

[0001] The present invention relates to the field of nano-biomedical photosensitivity diagnosis and treatment, and in particular to a cyanine near-infrared contrast agent and a preparation method and application thereof. Background Art

[0002] The complexity of tumorigenesis, high heterogeneity, potential drug resistance, and propensity for metastasis pose significant challenges to effective tumor treatment. Fluorescence imaging is an important imaging modality used in research and clinical applications to improve disease detection and guide surgery. Compared to radiographic imaging techniques, fluorescence imaging offers significant advantages in safety and resolution.

[0003] In fluorescence imaging, near-infrared fluorescence imaging has been a research focus in recent years. The first near-infrared region (NIR-I; 700–900 nm) has become a hot topic in the biomedical field due to its high sensitivity, rapid response, lack of harmful radiation, and low cost. Compared with fluorophores emitting at visible wavelengths, fluorophores emitting in the NIR-I region can achieve deeper penetration and better imaging quality. Furthermore, recent studies have demonstrated that fluorescence imaging in the second near-infrared region (NIR-II, 1000–1700 nm) can achieve better fluorescence image quality and signal-to-noise ratio (SBR) than that in the NIR-I region. In the field of biomedical imaging, the near-infrared II region (NIR-II) has achieved significant improvements in imaging temporal and spatial resolution (~20 ms and ~25 mm) and penetration depth (up to ~3 cm) due to reduced scattering, negligible tissue absorption, and weak low-light fluorescence effects. Consequently, a series of fluorescent probes and fluorescent dyes have been developed using the principles of fluorescence imaging for tumor treatment and prevention. In addition, many contrast agents have been used for fluorescence imaging, including various fluorescent protein genes (such as GFP, RFP, YFP, etc.), organic fluorescent dyes, fluorescent nanoparticles, quantum dots, etc. However, these traditional contrast agents have problems such as poor biocompatibility, low tumor targeting efficiency, insufficient NIR-II signal-to-noise ratio, and heavy metal toxicity. Therefore, there is an urgent need for a contrast agent that can meet the requirements of fluorescence imaging and has low toxicity. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the deficiencies and defects in the prior art, thereby providing a cyanine near-infrared contrast agent and a preparation method and application thereof.

[0005] In order to solve the above problems, the present invention provides a cyanine near-infrared contrast agent having an amphiphilic molecular structure, and the structural formula is as follows:

[0006]

[0007] The molecular weight of cyanine near-infrared contrast agents is ≥3500.

[0008] The present invention also provides a method for preparing the above-mentioned cyanine near-infrared contrast agent, comprising the following steps:

[0009] (1) In a light-proof environment, a near-infrared cyanine dye, 3-mercaptopropionic acid, and a solvent are mixed to obtain a sodium salt solution, nitrogen is introduced into the sodium salt solution, and triethylamine is added thereto. Under a protective atmosphere, a substitution reaction is carried out to obtain a compound of formula I;

[0010] The structural formula of near-infrared cyanine dyes is as follows:

[0011]

[0012] The structural formula of the compound of formula I is as follows:

[0013]

[0014] The synthetic route is as follows:

[0015]

[0016] (2) mixing the compound of formula I, 3-azidopropylamine, a solvent, and a condensation reagent, and adding N,N-diisopropylethylamine under a protective atmosphere to carry out an amidation reaction to obtain a compound of formula II;

[0017] The structural formula of the compound of formula II is as follows:

[0018]

[0019] The synthetic route is as follows:

[0020]

[0021] (3) mixing hyaluronic acid, propargylamine, a solvent, and a condensation reagent, and adding N,N-diisopropylethylamine under a protective atmosphere to carry out an amidation reaction to obtain a compound of formula III;

[0022] The structural formula of the compound of formula III is as follows:

[0023]

[0024] The synthetic route is as follows:

[0025]

[0026] (4) mixing the compound of formula II, the compound of formula III, and a solvent, adding a catalyst under a protective atmosphere, and performing a linking chemical reaction to obtain a cyanine near-infrared contrast agent;

[0027] The synthetic route is as follows:

[0028]

[0029] Preferably, in step (1), the ratio of near-infrared cyanine dye, 3-mercaptopropionic acid, triethylamine and solvent is 0.38-0.42 mM: 0.76-0.84 mM: 0.78-0.82 mM: 5-10 mL;

[0030] The solvent includes N,N-dimethylformamide.

[0031] Preferably, the time for introducing nitrogen in step (1) is 20-30 min, the temperature for the substitution reaction is 40-50° C., and the time for the substitution reaction is 5-7 h.

[0032] Preferably, in step (2), the ratio of the compound of formula I, 3-azidopropylamine, condensation reagent, N,N-diisopropylethylamine and solvent is 0.38-0.42 mM: 0.76-0.84 mM: 0.4-0.42 mM: 0.5-0.55 mM: 10 mL;

[0033] The condensation reagent includes 2-(7-azobenzotriazole)-N,N,N,N'-tetramethyluronium hexafluorophosphate, and the solvent includes N,N-dimethylformamide.

[0034] Preferably, the temperature of the amidation reaction in step (2) is 20-30° C., and the amidation reaction time is 5-7 h.

[0035] Preferably, in step (3), the ratio of hyaluronic acid, propargylamine, condensation reagent, N,N-diisopropylethylamine and solvent is 0.38-0.42 mM: 0.76-0.84 mM: 0.4-0.42 mM: 0.5-0.55 mM: 10 mL;

[0036] The condensation reagent includes 2-(7-azobenzotriazole)-N,N,N,N'-tetramethyluronium hexafluorophosphate, and the solvent includes N,N-dimethylformamide.

[0037] Preferably, in step (4), the mass volume ratio of the compound of formula II, the compound of formula III and the solvent is 340-350 mg: 1-1.5 g: 10 mL.

[0038] Preferably, in step (4), the solvent comprises acetonitrile and the catalyst comprises copper sulfate.

[0039] Preferably, the temperature of the linking chemical reaction in step (4) is 55-65° C., and the time of the linking chemical reaction is 22-26 h.

[0040] The present invention also provides application of the cyanine near-infrared contrast agent in biomedical imaging.

[0041] The technical solution of the present invention has the following advantages:

[0042] The cyanine class near-infrared contrast agent provided by the present invention has the molecular structure of amphiphilic type, and the hydrophobic fluorescent core of near-infrared cyanine class dye (IR806) is combined with hyaluronic acid hydrophilic shell, can ensure the stability of fluorescence, and nonspecific adsorption is reduced again by hydrophilic chain, reduce the clearance rate of reticuloendothelial system (RES), prolong blood circulation time.At the same time, hyaluronic acid and the specific binding of tumor cell surface CD44 receptor can guide contrast agent to preferentially gather in tumor tissue, improve the targeting to tumor tissue, reduce the toxicity of near-infrared cyanine class dye (IR806) due to free causing.In addition, higher tumor targeting improves the enrichment speed of contrast agent, makes contrast agent can be rapidly high-density enriched at tumor site, reduces the interference of background fluorescence, improves the imaging effect of signal-to-noise ratio and NIR-II.In addition, hyaluronic acid has preferably water-soluble, and after cyanine molecule is carried out water-soluble modification, makes cyanine molecule have good biocompatibility, reduces immune system recognition, avoids being attacked by antibody or complement system.

[0043] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the compound of formula I in Example 1;

[0045] Figure 2 is the absorption spectrum of the compound of formula I in Example 1;

[0046] Figure 3 is the fluorescence spectrum of the compound of formula I in Example 1;

[0047] Figure 4 is the H NMR spectrum of the compound of formula II in Example 1;

[0048] Figure 5 is the infrared spectrum of the compound of formula II in Example 1;

[0049] Figure 6 is the absorption spectrum of the cyanine near-infrared contrast agent in Example 1;

[0050] Figure 7 This is a schematic diagram showing the effect of the cyanine near-infrared contrast agent prepared in Example 1 on the survival rate of mouse breast cancer cells;

[0051] Figure 8 This is a schematic diagram showing the effect of the cyanine near-infrared contrast agent prepared in Example 1 on the survival rate of mouse embryonic fibroblasts;

[0052] Figure 9 This is the second window near-infrared cell fluorescence imaging image of the cyanine near-infrared contrast agent prepared in Example 1;

[0053] in, Figure 9 a in the figure is the fluorescence imaging image at 0h. Figure 9 b in the figure is the fluorescence imaging image of 6h, Figure 9 The c in the figure is the fluorescence imaging image at 12h. Figure 9 d in the figure is the 22h fluorescence imaging image;

[0054] Figure 10 Schematic diagram of the relationship between fluorescence intensity and time in the second window near-infrared cell fluorescence imaging image of the cyanine near-infrared contrast agent prepared in Example 1. DETAILED DESCRIPTION

[0055] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0056] The present invention provides a cyanine near-infrared contrast agent having an amphiphilic molecular structure, and the structural formula is as follows:

[0057]

[0058] The molecular weight of cyanine near-infrared contrast agents is ≥3500.

[0059] The present invention also provides a method for preparing the above-mentioned cyanine near-infrared contrast agent, comprising the following steps:

[0060] (1) In a light-proof environment, a near-infrared cyanine dye, 3-mercaptopropionic acid, and a solvent are mixed to obtain a sodium salt solution, nitrogen is introduced into the sodium salt solution, and triethylamine is added thereto. Under a protective atmosphere, a substitution reaction is carried out to obtain a compound of formula I;

[0061] (2) mixing the compound of formula I, 3-azidopropylamine, a solvent, and a condensation reagent, and adding N,N-diisopropylethylamine under a protective atmosphere to carry out an amidation reaction to obtain a compound of formula II;

[0062] (3) mixing hyaluronic acid, propargylamine, and a solvent, adding a catalyst, and performing an amidation reaction to obtain a compound of formula III;

[0063] (4) Mixing the compound of formula II, the compound of formula III, and a solvent, adding a catalyst under a protective atmosphere, and performing a linking chemical reaction to obtain a cyanine near-infrared contrast agent.

[0064] In some embodiments of the present invention, the near-infrared cyanine dye is 2-[2-[2-chloro-3-[2-[1,3-dihydro-3,3-dimethyl-1-(4-sulfonic acid butyl)-2H-indol-2-ylidene]-ethylidene]-1-cyclopenten-1-yl]-vinyl]-3,3-dimethyl-1-(4-sulfonic acid butyl)-3H-indole hydroxide inner salt sodium salt (IR806).

[0065] In some embodiments of the present invention, in step (1), the ratio of near-infrared cyanine dye, 3-mercaptopropionic acid, triethylamine and solvent is 0.38-0.42 mM: 0.76-0.84 mM: 0.78-0.82 mM: 5-10 mL; and the solvent comprises N,N-dimethylformamide.

[0066] In some embodiments of the present invention, the purpose of introducing nitrogen in step (1) is to remove oxygen in the solution, the time for introducing nitrogen is 20-30 minutes, the temperature for the substitution reaction is 40-50° C., and the time for the substitution reaction is 5-7 hours.

[0067] In some embodiments of the present invention, after the substitution reaction in step (1) is completed, the substitution product is spin-dried and purified to obtain a compound of formula I; the developing solvent for purification includes petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 10:1.

[0068] In some embodiments of the present invention, in step (2), the ratio of the compound of formula I, 3-azidopropylamine, condensation reagent, N,N-diisopropylethylamine and solvent is 0.38-0.42 mM: 0.76-0.84 mM: 0.4-0.42 mM: 0.5-0.55 mM: 10 mL; the condensation reagent includes 2-(7-azobenzotriazole)-N,N,N,N'-tetramethyluronium hexafluorophosphate, and the solvent includes N,N-dimethylformamide.

[0069] In some embodiments of the present invention, the temperature of the amidation reaction in step (2) is 20-30° C., and the amidation reaction time is 5-7 h.

[0070] In some embodiments of the present invention, after the amidation reaction in step (2) is completed, the reaction product is extracted and purified to obtain a compound of formula II. The extraction solvent includes ethyl acetate and sodium chloride solution, and the purification solvent includes petroleum ether and ethyl acetate, with the volume ratio of petroleum ether to ethyl acetate being 20:1.

[0071] In some embodiments of the present invention, the mass volume ratio of hyaluronic acid, propargylamine, condensation reagent, N,N-diisopropylethylamine and solvent in step (3) is 0.8-1.2 g:0.4-0.44 mg:155-165 mg:65-75 mg:10 mL; the condensation reagent includes 2-(7-azobenzotriazole)-N,N,N,N'-tetramethyluronium hexafluorophosphate, and the solvent includes N,N-dimethylformamide.

[0072] In some embodiments of the present invention, the temperature of the amidation reaction in step (3) is 20-30° C., and the duration of the amidation reaction is 5-7 h.

[0073] In some embodiments of the present invention, after the amidation reaction is completed in step (3), the reaction product is dialyzed and dried to obtain a compound of formula III. The dialysis separation process comprises: mixing the reaction system solution with water, placing it in a dialysis bag with a molecular weight cut-off of 3500, and dialyzing it with deionized water as a dialysate for 48 hours, during which the dialysate is replaced every 6 hours.

[0074] In some embodiments of the present invention, the mass volume ratio of the compound of formula II, the compound of formula III and the solvent in step (4) is (340-350) mg: (1-1.5) g: 10 mL.

[0075] In some embodiments of the present invention, the solvent in step (4) includes acetonitrile, and the catalyst includes copper sulfate. The role of copper sulfate is only to catalyze the reaction and not as a reactant. Therefore, its amount is not further limited and can be used in an amount familiar to those skilled in the art.

[0076] In some embodiments of the present invention, the temperature of the linking chemical reaction in step (4) is 55-65° C., and the time of the linking chemical reaction is 22-26 hours.

[0077] In some embodiments of the present invention, after the linking chemical reaction is completed in step (4), dialysis and drying are performed to obtain a cyanine near-infrared contrast agent. The dialysis separation process comprises: mixing the reaction system solution with water, placing it in a dialysis bag with a molecular weight cut-off of 3500, and dialyzing it with deionized water as the dialysate for 48 hours, during which the dialysate is replaced every 6 hours.

[0078] In some embodiments of the present invention, the protective atmosphere in steps (1)-(4) each independently comprises nitrogen.

[0079] The present invention also provides the use of the cyanine near-infrared contrast agent or the cyanine near-infrared contrast agent prepared by the above-mentioned preparation method of the cyanine near-infrared contrast agent in biomedical imaging.

[0080] In some embodiments of the invention, biomedical imaging comprises near-infrared second window fluorescence imaging.

[0081] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0082] Example 1

[0083] A method for preparing a cyanine near-infrared contrast agent comprises the following steps:

[0084] (1) In a light-proof environment, 0.4 mmol of a near-infrared cyanine dye, 0.8 mmol of 3-mercaptopropionic acid, and 5 mL of dimethylformamide were mixed to obtain a sodium salt solution. Nitrogen was introduced into the sodium salt solution. After nitrogen bubbling for 20 minutes, 0.8 mmol of triethylamine was added. The substitution reaction was carried out at 45° C. for 6 hours under a nitrogen atmosphere. The substitution reaction product was dried by rotary evaporation and then purified by chromatography (the volume ratio of petroleum ether to ethyl acetate as the developing solvent was 10:1) to obtain a compound of formula I.

[0085] (2) Under light-shielding conditions, 0.4 mmol of the compound of formula I, 0.4 mmol of 3-azidopropylamine, 10 mL of N,N-dimethylformamide, and 0.4 mmol of condensing agent 2-(7-azobenzotriazole)-N,N,N,N'-tetramethyluronium hexafluorophosphate were added to the reaction tube. Then, 0.4 mmol of N,N-diisopropylethylamine was added under nitrogen atmosphere. The amidation reaction was carried out at 25°C for 6 hours. After the reaction, the product was extracted with dichloromethane and water as the extraction solvent. The lower organic phase was taken and dried by spin drying, and then purified on a chromatographic column (developing solvent polarity is petroleum ether: ethyl acetate = 20:1) to obtain the compound of formula II.

[0086] (3) 1 g of hyaluronic acid, 0.42 mg of propargylamine, 10 mL of N,N-dimethylformamide, and 160 mg of condensing agent 2-(7-azobenzotriazole)-N,N,N,N'-tetramethyluronium hexafluorophosphate were taken, and then 70 mg of N,N-diisopropylethylamine was added under nitrogen atmosphere to carry out amidation reaction at 25°C for 6 hours. After the reaction, the solution in the product system was added to 20 mL of deionized water, and the obtained mixed aqueous solution was placed in a dialysis bag with a molecular weight cutoff of 3500 and dialyzed with deionized water as the dialysate for 48 hours. The dialysate was changed once every 6 hours. After the dialysis, the solution in the dialysis bag was freeze-dried to obtain the compound of formula III.

[0087] (4) Under light-shielding conditions, 345 mg of the compound of formula II, 1.2 g of the compound of formula III, and 10 mL of acetonitrile were mixed, and 0.5 g of copper sulfate was added under a nitrogen atmosphere. The reaction was carried out at 60° C. for 24 h. After the reaction, the solution in the product system was added to 20 mL of deionized water, and the resulting mixed aqueous solution was placed in a dialysis bag with a molecular weight cutoff of 3500. The solution was dialyzed with deionized water as the dialysate for 48 h, and the dialysate was changed every 6 h. After the dialysis, the solution in the dialysis bag was freeze-dried to obtain a cyanine near-infrared contrast agent.

[0088] Example 2

[0089] A method for preparing a cyanine near-infrared contrast agent comprises the following steps:

[0090] (1) In a light-proof environment, 0.38 mmol of a near-infrared cyanine dye, 0.76 mmol of 3-mercaptopropionic acid, and 10 mL of dimethylformamide were mixed to obtain a sodium salt solution. Nitrogen was introduced into the sodium salt solution. After nitrogen bubbling for 30 minutes, 0.78 mmol of triethylamine was added. The substitution reaction was carried out at 40° C. for 7 hours under a nitrogen atmosphere. The substitution reaction product was dried by rotary evaporation and then purified by chromatography (the volume ratio of petroleum ether to ethyl acetate as the developing solvent was 10:1) to obtain a compound of formula I.

[0091] (2) Under light-shielding conditions, 0.38 mmol of the compound of formula I, 0.76 mmol of 3-azidopropylamine, 10 mL of N,N-dimethylformamide, and 0.42 mmol of condensing agent 2-(7-azobenzotriazole)-N,N,N,N'-tetramethyluronium hexafluorophosphate were added to the reaction tube. Then, 0.5 mmol of N,N-diisopropylethylamine was added under nitrogen atmosphere. The amidation reaction was carried out at 20°C for 7 hours. After the reaction, the product was extracted with dichloromethane and water as the extraction solvent. The lower organic phase was taken and dried by spin drying, and then purified on a chromatography column (developing solvent polarity is petroleum ether: ethyl acetate = 20:1) to obtain the compound of formula II.

[0092] (3) 0.8 g of hyaluronic acid, 0.4 mg of propargylamine, 10 mL of N,N-dimethylformamide, and 150 mg of condensing agent 2-(7-azobenzotriazole)-N,N,N,N'-tetramethyluronium hexafluorophosphate were taken, and then 65 mg of N,N-diisopropylethylamine was added under nitrogen atmosphere to carry out amidation reaction at 25°C for 6 hours. After the reaction, the solution in the product system was added to 20 mL of deionized water, and the obtained mixed aqueous solution was placed in a dialysis bag with a molecular weight cutoff of 3500 and dialyzed with deionized water as the dialysate for 48 hours. The dialysate was changed once every 6 hours. After the dialysis, the solution in the dialysis bag was freeze-dried to obtain the compound of formula III.

[0093] (4) Under light-shielding conditions, 340 mg of the compound of formula II, 1 g of the compound of formula III, and 10 mL of acetonitrile were mixed, copper sulfate was added under a nitrogen atmosphere, and a chemical reaction was carried out at 55°C for 22 hours. After the reaction, the solution in the product system was added to 20 mL of deionized water, and the resulting mixed aqueous solution was placed in a dialysis bag with a molecular weight cutoff of 3500, and dialyzed with deionized water as the dialysate for 48 hours. During this period, the dialysate was changed once every 6 hours. After the dialysis, the solution in the dialysis bag was freeze-dried to obtain a cyanine near-infrared contrast agent.

[0094] Example 3

[0095] A method for preparing a cyanine near-infrared contrast agent comprises the following steps:

[0096] (1) In a light-proof environment, 0.42 mmol of a near-infrared cyanine dye, 0.84 mmol of 3-mercaptopropionic acid, and 5 mL of dimethylformamide were mixed to obtain a sodium salt solution. Nitrogen was introduced into the sodium salt solution. After nitrogen bubbling for 20 minutes, 0.82 mmol of triethylamine was added. The substitution reaction was carried out at 50° C. for 5 hours under a nitrogen atmosphere. The substitution reaction product was dried and then purified by chromatography (the volume ratio of petroleum ether to ethyl acetate as the developing solvent was 10:1) to obtain a compound of formula I;

[0097] (2) Under light-shielding conditions, 0.42 mmol of the compound of formula I, 0.84 mmol of 3-azidopropylamine, 10 mL of N,N-dimethylformamide, and 0.42 mmol of the condensing agent 2-(7-azobenzotriazole)-N,N,N,N'-tetramethyluronium hexafluorophosphate were added to the reaction tube. Then, 0.55 mmol of N,N-diisopropylethylamine was added under nitrogen atmosphere. The amidation reaction was carried out at 30°C for 5 hours. After the reaction, the product was extracted with dichloromethane and water as the extraction solvent. The lower organic phase was taken and dried by spin drying, and then purified on a chromatographic column (developing solvent polarity is petroleum ether: ethyl acetate = 20:1) to obtain the compound of formula II.

[0098] (3) 1.2 g of hyaluronic acid, 0.44 mg of propargylamine, 10 mL of N,N-dimethylformamide, and 165 mg of condensing agent 2-(7-azobenzotriazole)-N,N,N,N'-tetramethyluronium hexafluorophosphate were taken, and then 75 mg of N,N-diisopropylethylamine was added under nitrogen atmosphere to carry out amidation reaction at 25°C for 6 hours. After the reaction, the solution in the product system was added to 20 mL of deionized water, and the obtained mixed aqueous solution was placed in a dialysis bag with a molecular weight cutoff of 3500 and dialyzed with deionized water as the dialysate for 48 hours. The dialysate was changed once every 6 hours. After the dialysis, the solution in the dialysis bag was freeze-dried to obtain the compound of formula III.

[0099] (4) Under light-shielding conditions, 350 mg of the compound of formula II, 1.5 g of the compound of formula III, and 10 mL of acetonitrile were mixed, copper sulfate was added under a nitrogen atmosphere, and a chemical reaction was carried out at 65° C. for 26 h. After the reaction, the solution in the product system was added to 20 mL of deionized water, and the resulting mixed aqueous solution was placed in a dialysis bag with a molecular weight cutoff of 3500, and dialyzed with deionized water as the dialysate for 48 h. During this period, the dialysate was changed once every 6 h. After the dialysis, the solution in the dialysis bag was freeze-dried to obtain a cyanine near-infrared contrast agent.

[0100] Characterization testing

[0101] The compound of formula I obtained in step (1) of Example 1 was subjected to nuclear magnetic resonance detection, and an organic solution of the compound of formula I was prepared using dichloromethane as an organic solvent, and its absorption spectrum and fluorescence spectrum were tested. The results were as follows: Figure 1 、 Figure 2 and Figure 3 shown.

[0102] from Figure 1 The H NMR spectrum of the compound of formula I shows the characteristic proton signal with the same structure as the compound of formula I; Figure 2 The absorption spectrum shows that the absorption peak is located in the 806nm band; Figure 3 The fluorescence spectrum shows that its absorption peak is located in the 896 nm band. Figure 1-Figure 3 The results show that the compound of formula I was successfully prepared in Example 1.

[0103] The compound of formula II obtained in step (2) of Example 1 was subjected to nuclear magnetic resonance detection and infrared spectroscopy detection. The results are as follows: Figure 4 and Figure 5 shown.

[0104] from Figure 4 The H NMR spectrum shows characteristic proton signals identical to those of the compound of formula II. Figure 5 The infrared spectrum shows that at 2050cm -1 There is a peak characterized by N3. Figure 4 and Figure 5 The results show that Example 1 successfully prepared the compound of formula II.

[0105] The organic solution of the cyanine near-infrared contrast agent prepared in step (4) of Example 1 was prepared using N,N-dimethylformamide as an organic solvent, and its absorption spectrum was tested. Figure 6 shown.

[0106] from Figure 6 It can be seen that its absorption peak is located at 806 nm, which is consistent with the structural formula of the compound. This shows that Example 1 successfully prepared a cyanine near-infrared contrast agent that meets the structural formula.

[0107] Performance Testing

[0108] The cyanine near-infrared contrast agent prepared in Example 1 was prepared into a series of cell culture solutions with a certain concentration gradient, and then mixed with mouse embryonic fibroblasts. (3T3) Mouse breast cancer cells (4T1) were cultured for 4 hours, CCK-8 reagent was added, and the absorbance at 450 nm was measured. The absorbance obtained was converted into cell survival rate, such as Figure 7 and Figure 8 shown.

[0109] from Figure 7 It can be seen that after the addition of cyanine near-infrared contrast agents, the survival rate of mouse embryonic fibroblasts (normal cells) is higher, indicating that the cyanine near-infrared contrast agent prepared in Example 1 has low toxicity to mouse fibroblasts and good biocompatibility.

[0110] from Figure 8 It can be seen that after the addition of the cyanine near-infrared contrast agent, the survival rate of breast cancer cells in mice is higher, indicating that the cyanine near-infrared contrast agent prepared in Example 1 has low toxicity to mouse breast cancer cells and has good biocompatibility.

[0111] The cyanine near-infrared contrast agent prepared in Example 1 was prepared into a cell culture solution with a concentration of 1 mg / mL. The above culture solution was used to culture mouse breast cancer cells for 4 hours, and then excited with an 808 nm laser. Fluorescence images of the cells were taken in a second near-infrared window fluorescence imager, as shown in FIG. Figure 9 As shown. At the same time, the relationship between fluorescence intensity and time is measured, as shown Figure 10 shown.

[0112] from Figure 9 It can be seen that the cyanine near-infrared contrast agent prepared in Example 1 has the performance of near-infrared second-zone fluorescence imaging; Figure 10 It can be seen that the fluorescence intensity becomes stronger as time goes by.

[0113] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A cyanine near-infrared contrast agent, characterized in that: Cyanine near-infrared contrast agents have an amphiphilic molecular structure, and the structural formula is as follows: The molecular weight of cyanine near-infrared contrast agents is ≥3500.

2. A method for preparing the cyanine near-infrared contrast agent according to claim 1, characterized in that: The following steps are included: (1) In a light-proof environment, a near-infrared cyanine dye, 3-mercaptopropionic acid, and a solvent are mixed to obtain a sodium salt solution, nitrogen is introduced into the sodium salt solution, and triethylamine is added thereto. Under a protective atmosphere, a substitution reaction is carried out to obtain a compound of formula I; The structural formula of the compound of formula I is as follows: (2) mixing the compound of formula I, 3-azidopropylamine, a solvent, and a condensation reagent, and adding N,N-diisopropylethylamine under a protective atmosphere to carry out an amidation reaction to obtain a compound of formula II; The structural formula of the compound of formula II is as follows: (3) mixing hyaluronic acid, propargylamine, a solvent, and a condensation reagent, and adding N,N-diisopropylethylamine under a protective atmosphere to carry out an amidation reaction to obtain a compound of formula III; (4) Mixing the compound of formula II, the compound of formula III, and a solvent, adding a catalyst under a protective atmosphere, and performing a linking chemical reaction to obtain a cyanine near-infrared contrast agent.

3. The method for preparing a cyanine near-infrared contrast agent according to claim 2, characterized in that: In step (1), the ratio of the near-infrared cyanine dye, 3-mercaptopropionic acid, triethylamine, and solvent is 0.38-0.42 mM: 0.76-0.84 mM: 0.78-0.82 mM: 5-10 mL; The solvent includes N,N-dimethylformamide.

4. The method for preparing a cyanine near-infrared contrast agent according to claim 2, wherein: In step (1), the time for introducing nitrogen is 20-30 minutes, the temperature for the substitution reaction is 40-50° C., and the time for the substitution reaction is 5-7 hours.

5. The method for preparing a cyanine near-infrared contrast agent according to claim 2, characterized in that: In step (2), the ratio of the compound of formula I, 3-azidopropylamine, condensation reagent, N,N-diisopropylethylamine and solvent is 0.38-0.42 mM: 0.76-0.84 mM: 0.4-0.42 mM: 0.5-0.55 mM: 10 mL; In step (2), the condensation reagent includes 2-(7-azobenzotriazole)-N,N,N,N'-tetramethyluronium hexafluorophosphate, and the solvent includes N,N-dimethylformamide.

6. The method for preparing a cyanine near-infrared contrast agent according to claim 2, characterized in that: The temperature of the amidation reaction in step (2) is 20-30° C., and the time of the amidation reaction is 5-7 h.

7. The method for preparing a cyanine near-infrared contrast agent according to claim 2, characterized in that: In step (3), the ratio of hyaluronic acid, propargylamine, condensation reagent, N,N-diisopropylethylamine and solvent is 0.38-0.42 mM: 0.76-0.84 mM: 0.4-0.42 mM: 0.5-0.55 mM: 10 mL; In step (3), the condensation reagent includes 2-(7-azobenzotriazole)-N,N,N,N'-tetramethyluronium hexafluorophosphate, and the solvent includes N,N-dimethylformamide.

8. The method for preparing a cyanine near-infrared contrast agent according to claim 2, characterized in that: The temperature of the amidation reaction in step (3) is 20-30° C., and the time of the amidation reaction is 5-7 h.

9. The method for preparing a cyanine near-infrared contrast agent according to claim 2, characterized in that: In step (4), the ratio of the compound of formula II, the compound of formula III and the solvent is 340-350 mg: 1-1.5 g: 10 mL; The solvent includes acetonitrile, and the catalyst includes copper sulfate; The temperature of the linking chemical reaction in step (4) is 55-65° C., and the time of the linking chemical reaction is 22-26 h.

10. A use of the cyanine near-infrared contrast agent according to claim 1, characterized in that: Applications in biomedical imaging.

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