CN113683737B - A kind of polymer fluorescent probe, preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of fluorescent probes, in particular to a polymer fluorescent probe, a preparation method and applications thereof. The hydrophilic monomers and cyanine dye monomers are copolymerized into hydrophilic dye polymers by radical polymerization, and then the complexes of lanthanide elements are grafted through substitution reaction, and the energy transfer between lanthanide elements and cyanine dyes is carried out. It can realize the enhancement of tail peak emission of cyanine dyes in the second near-infrared region. The polymer fluorescent probe has good water solubility and biocompatibility, and has excellent near-infrared second-region fluorescence imaging effect.

Description

A kind of polymer fluorescent probe, preparation method and application thereof

technical field

The invention relates to the technical field of fluorescent probes, in particular to a polymer fluorescent probe, a preparation method and applications thereof.

Background technique

In recent years, near-infrared second-region optical window (NIR-II, 1000-1700 nm) fluorescence imaging has been widely studied and applied in biomedicine and other fields, such as liver function assessment, monitoring treatment response in inflammatory arthritis, non-specific Heterogeneous tumor targeting and image-mediated tumor therapy, etc.

Compared with the shorter wavelength visible light region (VIS, 400-650nm) and near-infrared region (NIR-I, 650-1000nm), near-infrared region II imaging can reduce biological tissue light scattering and reduce tissue autofluorescence, so it is extremely With greatly improved signal-to-noise ratio (SNR), imaging resolution and tissue penetration depth, the development of new near-infrared second-region fluorescent imaging materials has become a research hotspot. At present, near-infrared second-region fluorescent imaging materials can be divided into inorganic materials and organic materials, among which organic materials include organic small molecules and conjugated polymers. Inorganic materials usually contain heavy metal ions or are often coated with amphiphilic polymers, which are prone to leakage and separation when applied in vivo, and have non-negligible potential toxicity; small organic molecules have good biocompatibility and are easy to excrete in vivo. Such advantages have been applied to near-infrared second-region imaging. However, the currently available small organic molecules are prone to aggregation in aqueous solution, resulting in severe fluorescence quenching; conjugated polymers have attracted extensive attention due to their high molar extinction coefficient and easy preparation. The water solubility of fluorescent probes is poor; these shortcomings largely limit the application of fluorescent probes in near-infrared second-region bioimaging.

In view of the above-mentioned defects, the creator of the present invention finally obtained the present invention after a long period of research and practice.

SUMMARY OF THE INVENTION

The purpose of the present invention is to solve the problem that the organic small molecules in the near-infrared second-region fluorescent imaging materials are easy to aggregate in aqueous solution and cause serious fluorescence quenching, and the poor water solubility of conjugated polymer fluorescent probes limits the fluorescent probes in near The problem of application in infrared bioimaging in the second region provides a polymer fluorescent probe, a preparation method and its application.

In order to achieve the above purpose, the present invention discloses a preparation method of a polymer fluorescent probe, comprising the following steps:

S1: Preparation of cyanine dye monomers: A double bond-containing group is introduced into the cyanine dye through a substitution reaction, so that it can participate in free radical polymerization;

S2: preparing a dye polymer: by free radical polymerization, the cyanine dye monomer obtained in step S1 is copolymerized with a hydrophilic monomer to obtain a hydrophilic dye polymer;

S3: Preparation of lanthanide complex: dissolve the chelate and the salt of lanthanide in deionized water, heat up and adjust the pH to 7-8 and then stir to obtain a lanthanide complex;

S4: Preparation of polymer fluorescent probe: The lanthanide complex obtained in step S3 is introduced into the hydrophilic dye polymer obtained in step S2 through a substitution reaction, and the unreacted lanthanide complex is removed by dialysis, The polymer fluorescent probe is obtained by drying.

In the step S1, the cyanine dye is any one of IR-783, IR-808, ICG, IR-820 and derivatives of IR-783, IR-808, ICG and IR-820.

In the step S2, the hydrophilic monomers are poly(ethylene glycol) methacrylate with weight average molecular weight Mw=300, poly(ethylene glycol) methacrylate with weight average molecular weight Mw=360, methacrylic acid Any of hydroxyethyl ester and hydroxyethyl acrylate.

In the step S2, the molar ratio of the cyanine dye monomer and the hydrophilic monomer is 1:50-50000.

The average molecular weight of the cyanine dye polymer obtained in the step S2 is 2000-40000.

In the step S3, the chelating agent is any one of DTPA, DOTA, DO3A, NOTA, NTA, EDTA and derivatives of DTPA, DOTA, DO3A, NOTA, NTA and EDTA.

The salts of lanthanide elements in the step S3 are CeCl ₃ , NdCl ₃ , SmCl ₃ , EuCl ₃ , GdCl ₃ , TbCl ₃ , DyCl ₃ , HoCl ₃ , ErCl ₃ , TmCl ₃ , YbCl ₃ , CeCl ₃ , NdCl ₃ , Any of SmCl ₃ , EuCl ₃ , GdCl ₃ , TbCl ₃ , DyCl ₃ , HoCl ₃ , ErCl ₃ , TmCl ₃ , and YbCl ₃ hydrate.

In the step S4, the molar ratio of the dye polymer and the lanthanide complex is 1:1-500.

The invention also discloses a polymer fluorescent probe prepared by the above preparation method and the application of the polymer fluorescent probe in the field of near-infrared second region biological imaging.

The lanthanide complex-enhanced cyanine dyes prepared by the above preparation method are polymer fluorescent probes emitted in the near-infrared second region. Due to the large amount of hydrophilic segments, they dissolve in water to form a large number of hydrogen bonds. The cyanine dyes Tends to form assemblies with water-insoluble backbones, resulting in distorted intramolecular charge transfer, resulting in a red-shift in the emission of cyanine dyes. The cyanine dyes are fixed to the polymer chain due to the polymerization, which effectively reduces the aggregation quenching effect caused by the self-assembly of the cyanine dyes in water, and effectively improves the emission intensity of the cyanine dyes. Due to the 4f-4f energy level transition of lanthanides, the emission and absorption overlap with cyanine dyes, resulting in fluorescence resonance energy transfer between lanthanides and cyanine dyes. In addition, since the hydrophilic segments can rotate freely, the lanthanide complexes attached to the hydrophilic segments and the cyanine dyes emit fast intersystem interactions, resulting in triplet-triplet energy transfer. Compared with free cyanine dyes, these factors lead to the substantial enhancement of the tail peak emission of the polymer fluorescent probes in the near-infrared second region, which enables these cyanine dyes, which originally emit very weakly in the near-infrared second region. For near-infrared second-region fluorescence imaging.

Compared with the prior art, the beneficial effects of the present invention are:

(1) The present invention utilizes relatively low-cost cyanine dyes as raw materials, and achieves a very good near-infrared second-region fluorescence imaging effect. Compared with ordinary near-infrared second-region organic fluorophores, complicated organic synthesis steps are avoided;

(2) The polymer fluorescent probe of the present invention has good water solubility and biocompatibility;

(3) The polymer fluorescent probe of the present invention can adjust the fluorescence intensity by adjusting the types of lanthanide elements, so as to cope with different application scenarios.

Description of drawings

Fig. 1 is the ¹ H-NMR chart of the small molecule fluorescent dye monomer MA-IR-808 prepared in Example 1 of the present invention;

Fig. 2 is the ¹ H-NMR chart of the dye polymer P-OEGMA-Dye prepared in Example 1 of the present invention;

Figure 3 is the excitation of the polymer fluorescent probe P-OEGMA-Dye-Ln (Ln=Sm ³⁺ , Eu ³⁺ , Tb ³⁺ , Ho ³⁺ , Tm ³⁺ ) prepared in Examples 1-2 of the present invention at 808 nm The fluorescence emission spectrum under the graph;

Figure 4 is the excitation of the polymer fluorescent probe P-OEGMA-Dye-Ln (Ln=Sm ³⁺ , Eu ³⁺ , Tb ³⁺ , Ho ³⁺ , Tm ³⁺ ) prepared in Examples 1-2 of the present invention at 808 nm Fluorescence imaging picture under 1250nm filter;

Figure 5 shows that P-OEGMA-Dye-Ho and P-OEGMA-Dye obtained in Example 1 were injected into BLAB/c mice according to the effective content of IR-808 at 20 μg, and the images were observed under the near-infrared second-region fluorescence imager renderings.

Detailed ways

The above and other technical features and advantages of the present invention will be described in more detail below with reference to the accompanying drawings.

Example 1

In this example, the preparation of the small molecule fluorescent dye monomer is carried out according to the following steps:

S1: IR-808 (100 mg, 0.12 mmol) and the weighed N,N'-dicyclohexylcarbodiimide (50 mg, 0.24 mmol, DCC) and 2-aminoethyl methacrylate hydrochloride ( 100 mg, 0.6 mmol) was dissolved in 10 mL of N,N-dimethylformamide (DMF) and transferred to a polymerization flask connected to a Schlenk tube. Nitrogen protection was introduced to remove oxygen in the system, and the reaction was carried out in an oil bath at 40 °C for 24 h in the dark. After the reaction, the DMF was spin-dried and reconstituted with 10 mL of deionized water, and then the solution was transferred to a 1000D dialysis bag for dialysis in a water bath protected from light for 48 hours. After the dialysis, the water was spin-dried and vacuum-dried to obtain 95 mg of MA-IR-808 small molecule fluorescent dye monomer. FIG. 1 is a ¹ H-NMR chart of MA-IR-808.

S2: 4-cyano-4-(phenylthiocarbonylthio)valeric acid (70 mg, 0.25 mmol, CTA-COOH), polyethylene glycol methacrylate monomer (4.5 g, 12.5 mmol, OEGMA -OH, Mw=360), MA-IR-808 (12.5 mg, 0.0125 mmol) and azobisisobutyronitrile (8.2 mg, 0.05 mmol, AIBN) were dissolved in 20 mL of DMF and transferred to the polymerization connected to a Schlenk tube bottle. Under nitrogen protection and liquid nitrogen freezing conditions, oxygen was removed through a standard three freeze-thaw cycle process of freezing-nitrogen filling-deoxygenation-thawing. After the operation was completed, the polymerization was carried out in an oil bath at 70° C. in the dark for 12 h. After the reaction, the DMF was spin-dried and reconstituted with 10 mL of tetrahydrofuran (THF). Then, the solution was slowly added dropwise to 200 mL of n-hexane for precipitation, and vacuum-dried to obtain 4 g of P-OEGMA-Dye dye polymer. FIG. 2 is a ¹ H-NMR chart of P-OEGMA-Dye.

S3: Dissolve 2g HoCl ₃ ·6H ₂ O and 2g DTPA in 20 mL of deionized water, adjust the pH to 7-8 with 4M NaOH aqueous solution, and stir and react at 60°C for 12 h to obtain Ho-DTPA aqueous solution.

S4: Co-dissolve the Ho-DTPA aqueous solution with 1 g of P-OEGMA-Dye, add 2 g of EDC and 1 g of Hobt, and stir at 40 °C for 24 h. After the reaction, the insolubles were removed by centrifugation, and then dialyzed for 48 h. After fully drying, the P-OEGMA-Dye-Ho polymer fluorescent probe was obtained.

Example 2

S1: IR-808 (200 mg, 0.24 mmol) and weighed N,N'-dicyclohexylcarbodiimide (100 mg, 0.48 mmol, DCC) and 2-aminoethyl methacrylate hydrochloride ( 200 mg, 1.2 mmol) was dissolved in 20 mL of DMF and transferred to a polymerization flask connected to a Schlenk tube. Nitrogen protection was introduced to remove oxygen in the system, and the reaction was carried out in an oil bath at 40 °C for 24 h in the dark. After the reaction, the DMF was spin-dried and reconstituted with 20 mL of deionized water, and then the solution was transferred to a 1000D dialysis bag for dialysis in a water bath for 48 hours in the dark. After the dialysis, the water was spin-dried and vacuum-dried to obtain 200 mg of MA-IR-808 small molecule fluorescent dye monomer.

S2: 4-cyano-4-(phenylthiocarbonylthio)valeric acid (140 mg, 0.5 mmol, CTA-COOH), polyethylene glycol methacrylate monomer (9 g, 25 mmol, OEGMA-OH) , Mw=360), MA-IR-808 (25 mg, 0.025 mmol) and azobisisobutyronitrile (16 mg, 0.1 mmol, AIBN) were dissolved in 30 mL of DMF and transferred to a polymerization flask connected to a Schlenk tube. Under nitrogen protection and liquid nitrogen freezing conditions, oxygen was removed through a standard three freeze-thaw cycle process of freezing-nitrogen filling-deoxygenation-thawing. After the operation was completed, the polymerization was carried out in an oil bath at 70° C. in the dark for 12 h. After the reaction, the DMF was spin-dried and reconstituted with 20 mL of THF. Then, the solution was slowly added dropwise to 400 mL of n-hexane for precipitation, and vacuum dried to obtain 8 g of P-OEGMA-Dye dye polymer.

S3: Dissolve 2g SmCl ₃ ·6H ₂ O and 2g DTPA in 20 mL of deionized water, adjust the pH to 7-8 with 4M NaOH aqueous solution, stir and react at 60° C. for 12 h to obtain Sm-DTPA aqueous solution. Eu-DTPA, Tb-DTPA, Tm-DTPA were obtained in the same way.

S4: Co-dissolve the Sm-DTPA aqueous solution with 1 g of P-OEGMA-Dye, add 2 g of EDC and 1 g of Hobt, and stir at 40 °C for 24 h. After the reaction, the insolubles were removed by centrifugation, and then dialyzed for 48 h. P-OEGMA-Dye-Sm is obtained after sufficient drying. P-OEGMA-Dye-Eu, P-OEGMA-Dye-Tb and P-OEGMA-Dye-Tm were obtained in the same way.

P-OEGMA-Dye-Ho obtained in Example 1 and P-OEGMA-Dye-Sm, P-OEGMA-Dye-Eu, P-OEGMA-Dye-Tb, P-OEGMA-Dye-Tm obtained in Example 2 According to the effective concentration of IR-808, 10μg/mL was dissolved in deionized water, and the fluorescence emission spectrum was tested. The test conditions were: 808nm laser light source, 1W excitation light power, 3nm grating, and the fluorescence emission spectrum was shown in Figure 3.

P-OEGMA-Dye-Ho obtained in Example 1 and P-OEGMA-Dye-Sm, P-OEGMA-Dye-Eu, P-OEGMA-Dye-Tb, P-OEGMA-Dye-Tm obtained in Example 2 According to the effective concentration of IR-808, 10μg/mL was dissolved in deionized water, and the imaging effect was observed under the near-infrared second region fluorescence imager. The test conditions were: 808nm laser light source, 1W excitation light power, 50ms exposure time, 1250nm filter , the imaging effect is shown in Figure 4.

The P-OEGMA-Dye-Ho and P-OEGMA-Dye obtained in Example 1 were injected into BLAB/c mice according to the effective content of IR-808 at 20 μg, and the imaging effect was observed under the near-infrared second-region fluorescence imager. The conditions are: 808nm laser light source, 1W excitation light power, 50ms exposure time, 1250nm filter, and the imaging effect is shown in Figure 5.

The above descriptions are only preferred embodiments of the present invention, which are merely illustrative rather than limiting for the present invention. Those skilled in the art understand that many changes, modifications and even equivalents can be made within the spirit and scope defined by the claims of the present invention, but all fall within the protection scope of the present invention.

Claims (9)

1. a preparation method of a polymer fluorescent probe, is characterized in that, comprises the following steps: S1: Preparation of cyanine dye monomers: A double bond-containing group is introduced into the cyanine dye through a substitution reaction, so that it can participate in free radical polymerization; S2: Preparation of dye polymer: by radical polymerization, the cyanine dye monomer obtained in step S1 is copolymerized with a hydrophilic monomer to obtain a hydrophilic dye polymer, and the hydrophilic monomer is a weight average Any of poly(ethylene glycol) methacrylate with molecular weight Mw=300, poly(ethylene glycol) methacrylate with weight average molecular weight Mw=360, hydroxyethyl methacrylate, and hydroxyethyl acrylate ; S3: Preparation of lanthanide complex: dissolving the chelate and the salt of lanthanide in deionized water, heating up and adjusting the pH to 7-8 and stirring to obtain a lanthanide complex; S4: Preparation of polymer fluorescent probe: The lanthanide complex obtained in step S3 is introduced into the hydrophilic dye polymer obtained in step S2 through a substitution reaction, and the unreacted lanthanide complex is removed by dialysis, The polymer fluorescent probe is obtained by drying. 2. The method for preparing a polymer fluorescent probe according to claim 1, wherein the cyanine dyes in the step S1 are IR-783, IR-808, ICG, IR-820 and IR-783 , any of IR-808, ICG and IR-820 derivatives. 3 . The method for preparing a polymer fluorescent probe according to claim 1 , wherein the molar ratio of the cyanine dye monomer and the hydrophilic monomer in the step S2 is 1:50-50000. 4 . 4 . The method for preparing a polymer fluorescent probe according to claim 1 , wherein the average molecular weight of the cyanine dye polymer obtained in the step S2 is 2000-40000. 5 . 5. the preparation method of a kind of polymer fluorescent probe as claimed in claim 1 is characterized in that, in described step S3, chelate is DTPA, DOTA, DO3A, NOTA, NTA, EDTA and DTPA, DOTA, DO3A , any of NOTA, NTA and EDTA derivatives. 6 . The method for preparing a polymer fluorescent probe according to claim 1 , wherein in the step S3, the salts of the lanthanide elements are CeCl ₃ , NdCl ₃ , SmCl ₃ , EuCl ₃ , GdCl ₃ , TbCl ₃ , DyCl ₃ , HoCl 3 , ErCl ₃ , TmCl ₃ , YbCl ₃ and CeCl ₃ , NdCl ₃ , SmCl ₃ , EuCl ₃ , GdCl ₃ , TbCl ₃ , DyCl ₃ , HoCl ₃ , ErCl ₃ , _{TmCl 3} , YbCl ₃ Any of the hydrates. 7 . The method for preparing a polymer fluorescent probe according to claim 1 , wherein the molar ratio of the dye polymer and the lanthanide complex in the step S4 is 1:1˜500. 8 . 8. A polymer fluorescent probe prepared by the preparation method according to any one of claims 1 to 7. 9 . The application of the polymer fluorescent probe according to claim 8 in the field of near-infrared second region biological imaging. 10 .

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