CN118063708A - Saccharide modified water-soluble conjugated polymer contrast agent, preparation method and application thereof - Google Patents

Abstract

The invention discloses a saccharide modified water-soluble conjugated polymer contrast agent, a preparation method and application thereof, wherein the contrast agent has a chemical structure shown in a formula III: The contrast agent has an amphiphilic molecular structure, comprises a polymer main chain and a side chain, wherein the polymer main chain has a donor and acceptor structure, can prolong the bond length in a conjugated framework, enable absorption to be red-shifted, improve the fluorescence intensity of the polymer, and improve living body contrast resolution and contrast; the side chains select common branched chain amino acids in the three proteins to provide nutrient substances required by abnormal proliferation of tumors, and the branched chain amino acids are connected to the side ends of the polymers through covalent bonds, so that a large amount of amino groups can be provided, the near infrared two-region conjugated polymer has good water solubility, more importantly, tumor targeting can be realized, cytotoxicity can be further reduced, the uptake degree of cells to materials can be enhanced, the enrichment rate of the materials can be improved, and the fluorescence imaging effect can be further improved.

Description

Saccharide modified water-soluble conjugated polymer contrast agent, preparation method and application thereof

Technical Field

The invention relates to the technical field of nano biomedical photosensitive diagnosis and treatment, in particular to a saccharide-modified water-soluble conjugated polymer contrast agent, a preparation method and application thereof.

Background

Malignant tumor is also called cancer, is one of the greatest threats facing human beings at present, and is the first three diseases which lead to death of human beings due to diseases together with heart diseases and cerebrovascular diseases. Malignant tumors have high mortality due to the characteristics of various types, high complexity, hidden early symptoms and the like. Therefore, early diagnosis and treatment of cancer have become a popular research direction.

In recent years, various imaging techniques, including Computed Tomography (CT), magnetic Resonance Imaging (MRI), and Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT), have been used for in vivo imaging. However, simultaneous in-situ, real-time imaging of multiple events in a living body is difficult to achieve due to insufficient spatial and temporal resolution and safety concerns. Fluorescence imaging of the second near infrared window (NIR-II, 1000-1700 nm) is an emerging technology with deeper tissue penetration, higher spatial resolution and higher temporal resolution than traditional fluorescence imaging (400-900 nm) because it reduces photon absorption and scattering and ignores tissue autofluorescence.

In recent years, photosensitive therapy has been attracting attention because of its minimally invasive and selective characteristics in treating tumors. The new therapeutic method for killing tumor cells by stimulating photosensitive material to kill tumor cells through light source, especially near infrared light source, is a combined therapy of photothermal therapy and photodynamic therapy. Most of the photosensitive treatment research is directed to improving the therapeutic effect of the photosensitizer, rather than improving the enrichment efficiency of the photosensitive treatment, so as to achieve low-dose accurate targeted treatment. Traditional targeting strategies include passive targeting, which promotes selective aggregation in tumors by modulating nanoparticle size and surface chemistry, enhancing Permeability and Retention (EPR) effects, and active targeting, which has the disadvantages of limited enrichment and high particle size requirements. Active targeting is through binding of high affinity ligands including polypeptides, proteins, nucleic acid aptamers, etc. to specific surface molecules that are predominantly expressed by cancer cells or tumor epithelial cells, with the disadvantage of relatively high costs for polypeptide proteins, etc. The key point of the improvement of the enrichment efficiency is to improve the specific targeting of the photosensitive material. Traditional photosensitive treatment is characterized in that a photosensitive material is coated in polyethylene glycol (PEG) to enter cells, tumor is targeted by EPR effect, and the polyethylene glycol with neutral water-soluble groups has no specific recognition capability with other molecules or structures, so that the enrichment degree is limited.

Currently clinically approved organic near infrared dyes are only two types of Methylene Blue (MB) and indocyanine green (ICG), and both dyes are small molecules and can be rapidly discharged from the body. However, their fluorescence emission is in the NIR-I region and has limited penetration depth for in vivo imaging. To date, only a few organic molecules, which are highly hydrophobic, water-insoluble dyes, have fluorescent emissions in the NIR-II region, which must be encapsulated in a polymer matrix for bioimaging, increasing the particle size beyond the renal filtration threshold (ca.40 kD). Recently, small molecule CH1055 (8.9 kDa) NIR-II organic dyes emit at about 1055nm under 808nm excitation, with high water solubility. Although this small molecule dye has shown promise in the preclinical, the study of NIR-II organic dyes with simple preparation process, high quantum yield, and high resolution imaging quality is still in the beginning.

Disclosure of Invention

The invention aims to solve the technical problem of providing a saccharide-modified water-soluble conjugated polymer contrast agent, a preparation method and application thereof, aiming at the defects in the prior art.

In order to solve the technical problems, the invention adopts the following technical scheme: in a first aspect of the present invention, there is provided a saccharide-modified water-soluble conjugated polymer contrast agent having a chemical structure represented by formula III:

Wherein x=y=4 to 8, and m=30 to 50.

In a second aspect of the present invention, there is provided a method for preparing a saccharide-modified water-soluble conjugated polymer contrast agent as described above, comprising the steps of:

s1, preparing a polymer 1 with a structure shown in the following formula I:

wherein x=y=4 to 8;

s2, carrying out atom transfer radical polymerization reaction on the polymer 1 and acrylic acid-N-succinimidyl ester to prepare a polymer 2 with a structure shown in the following formula II:

Wherein m=30 to 50;

S3, removing succinimide ester from the polymer 2, and carrying out amidation reaction on the polymer 2 and amino groups of mannose to prepare the polymer 3, namely the saccharide modified water-soluble conjugated polymer contrast agent, wherein the structural formula of the polymer is shown in a formula III.

Preferably, in step S1, the polymer 1 is prepared by performing a steller coupling reaction on a monomer 1 having a structure shown in IV, a monomer 2 having a structure shown in formula V, and a monomer 3 having a structure shown in VI, and the synthetic route is as follows:

Preferably, the step S1 specifically includes: and under the dark environment, adding the monomer 1, the monomer 2 and the monomer 3 into a solvent to be completely dissolved, then introducing nitrogen into the obtained solution to bubble for more than 20 minutes, adding a palladium catalyst into the solution, reacting for 2-4 hours at the temperature of 80-90 ℃ under the protection of nitrogen, filtering the product after the reaction is finished, and then settling in methanol to obtain the polymer 1.

Preferably, in the step S1, monomer 1: monomer 2: the feeding mole ratio of the monomer 3 is 1:2:1.

Preferably, the step S1 specifically includes: under the dark environment, 0.1mM of monomer 1, 0.2mM of monomer 2 and 0.1mM of monomer 3 are added into 1mL of toluene to be completely dissolved, then nitrogen is introduced into the obtained solution to be bubbled for more than 20 minutes, 50mg of bis (triphenylphosphine) palladium (II) dichloride is added into the solution, the reaction is carried out for 3 hours under the protection of nitrogen at 80 ℃, and after the reaction is finished, the product is filtered and then repeatedly settled in methanol, so that the polymer 1 is obtained.

Preferably, the step S2 specifically includes:

adding a polymer 1 and acrylic acid-N-succinimidyl ester into an organic solvent under the condition of avoiding light, then adding a catalyst, adding pentamethyldiethylenetriamine under the protection of nitrogen, reacting the obtained mixed solution for 9-12 hours at 80-100 ℃, filtering a product after the reaction is finished, and then repeatedly settling in diethyl ether to obtain a polymer 2; the synthetic route is as follows:

Preferably, in step S2, the molar ratio of polymer 1 to N-succinimidyl acrylate is 1:50-200.

Preferably, the step S2 specifically includes:

100mg of polymer 1, 887mg of N-succinimidyl acrylate are added to 1mL of anisole under the protection of light, then a catalyst CuBr is added, 50 mu L of pentamethyldiethylenetriamine is added under the protection of nitrogen, the obtained mixed solution is reacted for 12 hours at 90 ℃, and after the reaction is finished, the product is filtered, and then repeated sedimentation is carried out in diethyl ether, so that polymer 2 is obtained.

Preferably, the step S3 specifically includes:

Under the condition of avoiding light, adding polymer 2 and mannite hydrochloride into DMF for dissolution, adding triethylamine under the protection of nitrogen, and stirring and reacting for 24-60 hours at 35-45 ℃; adding the solution in the product system into deionized water after the reaction is finished, placing the obtained mixed aqueous solution into a dialysis bag, dialyzing with deionized water, and drying the solution in the dialysis bag after the dialysis is finished to obtain a polymer 3 with a chemical structure shown in a formula III, wherein the synthetic route is as follows:

preferably, the step S3 specifically includes:

Under the condition of avoiding light, 300mg of polymer 2 and 1.75mmol of mannosamine hydrochloride are placed in a round bottom flask, 5mL of DMF is added for dissolution, 1.75mmol of triethylamine is added under the protection of nitrogen, and stirring reaction is carried out for 48 hours at 40 ℃; after the reaction is finished, adding the solution in the product system into 20mL of deionized water, placing the obtained mixed aqueous solution into a dialysis bag with molecular weight cut-off of 3500, dialyzing with deionized water for 48 hours, changing the dialyzate for 1 time every 6 hours, and freeze-drying the solution in the dialysis bag after the dialysis is finished to obtain the polymer 3.

In a third aspect of the invention, there is provided the use of a saccharide-modified water-soluble conjugated polymer contrast agent as described above in near infrared second window fluorescence imaging.

The beneficial effects of the invention are as follows:

The invention provides a saccharide-modified water-soluble conjugated polymer contrast agent, a preparation method and application thereof, wherein the saccharide-modified water-soluble conjugated polymer contrast agent has an amphiphilic molecular structure and comprises a polymer main chain and a side chain, wherein the polymer main chain has a donor and acceptor structure, so that the length of a conjugated bond in a conjugated framework can be prolonged, the absorption is red-shifted, the fluorescence intensity of the polymer is improved, and the living body contrast resolution and contrast are improved; the side chains select common branched chain amino acids in the three proteins to provide nutrient substances required by abnormal proliferation of tumors, and the branched chain amino acids are connected to the side ends of the polymers through covalent bonds, so that a large amount of amino groups can be provided, the near infrared two-region conjugated polymer has good water solubility, more importantly, tumor targeting can be realized, cytotoxicity can be further reduced, the uptake degree of cells to materials can be enhanced, the enrichment rate of the materials can be improved, and the fluorescence imaging effect can be further improved. The polymer has the advantages of improving focus targeting enrichment, signal to noise ratio, imaging definition and the like when being used for fluorescence imaging, so that the polymer can be well applied to near infrared second window fluorescence imaging.

Drawings

FIG. 1 is an absorption spectrum of a polymer 1 prepared in example 1;

FIG. 2 is an absorption spectrum of the polymer 2 prepared in example 1;

FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of the polymer 3 produced in example 1;

FIG. 4 is a transmission electron micrograph of Polymer 3 obtained in example 1;

FIG. 5 is a schematic diagram showing the measured nano-diameters of dynamic light scattering of polymer 3 prepared in example 1;

FIG. 6 is a graph showing absorption and emission spectra of the polymer 3 prepared in example 1 at a concentration of 0.5 mg/mL;

FIG. 7 is a second window near infrared fluorescence imaging of Polymer 3 prepared in example 1;

FIG. 8 is a graph showing cell viability of the polymer 3 prepared in example 1 with respect to cytotoxicity of mouse breast cancer cells.

Detailed Description

The present invention is described in further detail below with reference to examples to enable those skilled in the art to practice the same by referring to the description.

It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

The test methods used in the following examples are conventional methods unless otherwise specified. The material reagents and the like used in the following examples are commercially available unless otherwise specified. The following examples were conducted under conventional conditions or conditions recommended by the manufacturer, without specifying the specific conditions. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1

A saccharide-modified water-soluble conjugated polymer contrast agent, the preparation method comprises the following steps:

S1, preparing a polymer I with a chemical structure shown in the following formula I through the following synthetic route:

The method comprises the following specific steps:

In a dark environment, an acceptor molecule (monomer 1, structure formula IV) 3, 6-bis (5-bromothiophen-2-yl) -2, 5-bis (2-hexyldecyl) pyrrolo [3,4-c ] pyrrole-1, 4 (2H, 5H) -dione (66.8 mg,0.1 mm), a donor molecule (monomer 2, structure formula V) 2, 6-bis (trimethyltin) -4, 8-bis (5- (2-ethylhexyl) thiophen-2-yl) -benzodithiophene (180.9 mg,0.2 mm), a donor molecule (monomer 3, structure formula VI) (2, 7-dibromo-9H-fluorene-9, 9-diyl) bis (propane-3, 1-diyl) bis (2-bromo-2-methylpropionate) (73.8 mg,0.1 mm) were added to a reaction tube, and after dissolving the solid with toluene (1 mL), nitrogen was bubbled for 20 minutes or more; bis (triphenylphosphine) palladium (II) dichloride (50 mg) was rapidly added to the flask, and the flask was allowed to react under nitrogen at 80 ℃ for 3 hours; the crude product was filtered through an organic filter to remove the solid catalyst, followed by repeated sedimentation in methanol to give a reddish brown polymer 1 (100 mg).

S2, performing atom transfer radical polymerization reaction on the polymer 1 and acrylic acid-N-succinimidyl ester to prepare a polymer 2 with a chemical structure shown in a formula II, wherein the synthetic route is shown as follows:

The method comprises the following specific steps:

Polymer 1 (100 mg), acrylic acid-N-succinimidyl ester (887 mg,5.24 mM) was added to a reaction tube under a dark condition, the above solid was dissolved with anisole (1 mL), cuBr was added as a catalyst, 50. Mu.L of pentamethyldiethylenetriamine was added as a ligand under a nitrogen atmosphere, the resulting mixed solution was reacted at 90℃for 12 hours, after the completion of the reaction, the solid catalyst was removed by filtration with an organic filter membrane, and then, the polymer 2 (900 mg) was repeatedly precipitated in diethyl ether to obtain a reddish-brown acrylic acid-N-succinimidyl ester side chain-substituted conjugated main chain polymer.

S3, removing succinimide ester from the polymer 2, and carrying out amidation reaction with mannite hydrochloride to prepare a polymer 3, wherein the structural formula is shown in a formula III, namely, a saccharide modified water-soluble conjugated polymer contrast agent, and the synthetic route is as follows:

The method comprises the following specific steps:

Under the condition of avoiding light, 300mg of polymer 2 and 1.75mmol of mannosamine hydrochloride are placed in a round bottom flask, 5mL of DMF is added for dissolution, 1.75mmol of triethylamine is added under the protection of nitrogen, and stirring reaction is carried out for 48 hours at 40 ℃; after the reaction is finished, adding the solution in the product system into 20mL of deionized water, placing the obtained mixed aqueous solution into a dialysis bag with molecular weight cut-off of 3500, dialyzing with deionized water for 48 hours, changing the dialyzate for 1 time every 6 hours, and freeze-drying the solution in the dialysis bag to remove water after the dialysis is finished, thereby obtaining the polymer 3.

The intermediate product, the final product prepared in example 1 were subjected to the following performance tests and characterization:

1. The organic solution of the polymer 1 prepared in example 1 was prepared by using methylene chloride as an organic solvent, and the absorption spectrum was tested, and as shown in fig. 1, it can be seen that the absorption peak was located in the 670 nm band.

2. The organic solution of the polymer 2 prepared in example 1 was prepared using methylene chloride as an organic solvent, and the absorption spectrum was tested, and as shown in fig. 2, it can be seen that the absorption peak was located at 668 nm.

3. Polymer 3 prepared in example 1 was subjected to nuclear magnetic resonance spectroscopy to obtain a nuclear magnetic resonance hydrogen spectrum of polymer 3, and as shown in FIG. 3, a characteristic proton signal of mannose side chains was seen.

4. Polymer 3 prepared in example 2 was formulated as a dilute strength aqueous solution and then subjected to the following test:

i) The aqueous solutions were analyzed by a transmission electron microscope, and the obtained transmission electron microscope image was shown in FIG. 4, and it was found from FIG. 4 that the particle size of the polymer 3 was less than 100 nm.

Ii) based on the formulated aqueous polymer 3 solution, measured according to dynamic light scattering: the average hydrodynamic diameter of polymer 3 in water was 65 nm and the resulting hydrodynamic number distribution is shown in FIG. 5.

5. Polymer 3 prepared in example 1 was formulated as an aqueous solution at a concentration of 0.5mg/mL, and the absorption and emission spectra of Polymer 3 were measured, and the results are shown in FIG. 6, as can be seen: the absorption peak of polymer 3 is in the 743 nm band and the emission peak is in the 1066 nm band under 808 nm laser excitation.

6. Polymer 3 prepared in example 1 was formulated as an aqueous solution with a concentration gradient of 1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, excited with 808 nm laser, and a fluorescence image of the solution was taken in a second near infrared window fluorescence imager, and the results are shown in FIG. 7, which demonstrate that Polymer 3 has near infrared two-zone fluorescence imaging properties and has light stability.

7. Preparing a series of cell culture solutions with a concentration gradient from the polymer 3 prepared in the example 1, wherein the highest concentration of the polymer in the cell culture solution is 0.5mg/mL; the above culture solution was used to culture with mouse breast cancer cells for 4 hours, CCK-8 reagent was added, and absorbance at 450 nm was measured, and the obtained absorbance was converted into cell viability, as shown in FIG. 8, as can be seen: the cytotoxicity of the polymer 3 is very low, which indicates that the saccharide-modified water-soluble conjugated polymer contrast agent prepared by the invention has good biocompatibility.

Although embodiments of the present invention have been disclosed above, it is not limited to the use of the description and embodiments, it is well suited to various fields of use for the invention, and further modifications may be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the particular details without departing from the general concepts defined in the claims and the equivalents thereof.

Claims (12)

1. A saccharide-modified water-soluble conjugated polymer contrast agent, which is characterized by having a chemical structure shown in a formula III:

Wherein x=y=4 to 8, and m=30 to 50.

2. A method for preparing the saccharide-modified water-soluble conjugated polymer contrast agent according to claim 1, comprising the steps of:

s1, preparing a polymer 1 with a structure shown in the following formula I:

wherein x=y=4 to 8;

s2, carrying out atom transfer radical polymerization reaction on the polymer 1 and acrylic acid-N-succinimidyl ester to prepare a polymer 2 with a structure shown in the following formula II:

Wherein m=30 to 50;

S3, removing succinimide ester from the polymer 2, and carrying out amidation reaction on the polymer 2 and amino groups of mannose to prepare the polymer 3, namely the saccharide modified water-soluble conjugated polymer contrast agent, wherein the structural formula of the polymer is shown in a formula III.

3. The method for preparing a saccharide-modified water-soluble conjugated polymer contrast agent according to claim 2, wherein in step S1, the polymer 1 is prepared by carrying out a steller coupling reaction on a monomer 1 having a structure shown in IV, a monomer 2 having a structure shown in formula V, and a monomer 3 having a structure shown in VI, and the synthetic route is as follows:

4. The method for preparing a saccharide-modified water-soluble conjugated polymer contrast agent according to claim 3, wherein the step S1 specifically comprises: and under the dark environment, adding the monomer 1, the monomer 2 and the monomer 3 into a solvent to be completely dissolved, then introducing nitrogen into the obtained solution to bubble for more than 20 minutes, adding a palladium catalyst into the solution, reacting for 2-4 hours at the temperature of 80-90 ℃ under the protection of nitrogen, filtering the product after the reaction is finished, and then settling in methanol to obtain the polymer 1.

5. The method for preparing a saccharide-modified water-soluble conjugated polymer contrast agent according to claim 4, wherein in the step S1, monomer 1: monomer 2: the feeding mole ratio of the monomer 3 is 1:2:1.

6. The method for preparing a saccharide-modified water-soluble conjugated polymer contrast agent according to claim 5, wherein the step S1 specifically comprises: under the dark environment, 0.1mM of monomer 1, 0.2mM of monomer 2 and 0.1mM of monomer 3 are added into 1mL of toluene to be completely dissolved, then nitrogen is introduced into the obtained solution to be bubbled for more than 20 minutes, 50mg of bis (triphenylphosphine) palladium (II) dichloride is added into the solution, the reaction is carried out for 3 hours under the protection of nitrogen at 80 ℃, and after the reaction is finished, the product is filtered and then repeatedly settled in methanol, so that the polymer 1 is obtained.

7. The method for preparing a saccharide-modified water-soluble conjugated polymer contrast agent according to claim 2, wherein the step S2 specifically comprises:

adding a polymer 1 and acrylic acid-N-succinimidyl ester into an organic solvent under the condition of avoiding light, then adding a catalyst, adding pentamethyldiethylenetriamine under the protection of nitrogen, reacting the obtained mixed solution for 9-12 hours at 80-100 ℃, filtering a product after the reaction is finished, and then repeatedly settling in diethyl ether to obtain a polymer 2; the synthetic route is as follows:

8. the method for producing a saccharide-modified water-soluble conjugated polymer contrast agent according to claim 7, wherein in step S2, the molar ratio of the polymer 1 to the N-succinimidyl acrylate is 1:50 to 200.

9. The method for preparing a saccharide-modified water-soluble conjugated polymer contrast agent according to claim 8, wherein the step S2 specifically comprises:

100mg of polymer 1, 887mg of N-succinimidyl acrylate are added to 1mL of anisole under the protection of light, then a catalyst CuBr is added, 50 mu L of pentamethyldiethylenetriamine is added under the protection of nitrogen, the obtained mixed solution is reacted for 12 hours at 90 ℃, and after the reaction is finished, the product is filtered, and then repeated sedimentation is carried out in diethyl ether, so that polymer 2 is obtained.

10. The method for preparing a saccharide-modified water-soluble conjugated polymer contrast agent according to claim 2, wherein the step S3 specifically comprises:

Under the condition of avoiding light, adding polymer 2 and mannite hydrochloride into DMF for dissolution, adding triethylamine under the protection of nitrogen, and stirring and reacting for 24-60 hours at 35-45 ℃; adding the solution in the product system into deionized water after the reaction is finished, placing the obtained mixed aqueous solution into a dialysis bag, dialyzing with deionized water, and drying the solution in the dialysis bag after the dialysis is finished to obtain a polymer 3 with a chemical structure shown in a formula III, wherein the synthetic route is as follows:

11. The method for preparing a saccharide-modified water-soluble conjugated polymer contrast agent according to claim 10, wherein the step S3 specifically comprises:

Under the condition of avoiding light, 300mg of polymer 2 and 1.75mmol of mannosamine hydrochloride are placed in a round bottom flask, 5mL of DMF is added for dissolution, 1.75mmol of triethylamine is added under the protection of nitrogen, and stirring reaction is carried out for 48 hours at 40 ℃; after the reaction is finished, adding the solution in the product system into 20mL of deionized water, placing the obtained mixed aqueous solution into a dialysis bag with molecular weight cut-off of 3500, dialyzing with deionized water for 48 hours, changing the dialyzate for 1 time every 6 hours, and freeze-drying the solution in the dialysis bag after the dialysis is finished to obtain the polymer 3.

12. Use of a saccharide-modified water-soluble conjugated polymer contrast agent according to claim 1 or prepared by the method according to any one of claims 2 to 11 in near infrared second window fluorescence imaging.