CN116769174A - Water-soluble conjugated polymer contrast agent, preparation method and application thereof - Google Patents
Water-soluble conjugated polymer contrast agent, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a water-soluble conjugated polymer contrast agent with near infrared second window for absorbing fluorescence, a preparation method and application thereof, wherein the water-soluble conjugated polymer contrast agent has a chemical structure shown in the following formula III:the water-soluble conjugated polymer contrast agent provided by the invention has an amphiphilic molecular structure and contains a polymerThe polymer main chain has a donor and acceptor structure, and can prolong the length of a conjugated bond in a conjugated framework, so that the absorption is red-shifted, the fluorescence intensity of the polymer is improved, and the living body contrast resolution and contrast are improved; the PEG molecules with amino groups are selected as side chains, and are connected through covalent bonds, so that the near infrared two-region conjugated polymer has good water solubility, and tumor enrichment is realized through enhancing permeability and retention effect, so that the effect of fluorescence imaging is realized.
Description
Technical Field
The invention relates to the field of nano biomedical materials, in particular to a water-soluble conjugated polymer contrast agent with near infrared second window absorption fluorescence, 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. Thus, early diagnosis of cancer has 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.
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 region, which must be encapsulated in a polymer matrix for bioimaging, increasing the particle size beyond the renal filtration threshold (ca.40 kD). The small molecule CH1055 (8.9 kDa) NIR two-domain organic dye emits at about 1055nm under 808nm excitation and has high water solubility. Although the micromolecular dye has shown prospect in clinic, the research of the near infrared two-region organic dye with simple preparation process, high quantum yield and high resolution imaging quality is still in the beginning, and more reliable schemes need to be provided.
Disclosure of Invention
The invention aims to solve the technical problem of providing a water-soluble conjugated polymer contrast agent with a near infrared second window for absorbing fluorescence, and a preparation method and application thereof.
In order to solve the technical problems, the invention adopts the following technical scheme: a water-soluble conjugated polymer contrast agent with a near infrared second window for absorbing fluorescence, which has a chemical structure shown in the following formula III:
formula III
Wherein x=y=4 to 8, and m=30 to 50.
The 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 conjugated bond length in the conjugated skeleton can be prolonged, the absorption is red-shifted, the fluorescence intensity of the polymer is improved, and the living body contrast resolution ratio and contrast ratio are improved; the PEG molecules with amino groups are selected as side chains, and are connected through covalent bonds, so that the near infrared two-region conjugated polymer has good water solubility, and tumor enrichment is realized through Enhancing Permeability and Retention (EPR) effect, so that the effect of fluorescence imaging is realized. The above characteristics of the water-soluble conjugated polymer contrast agent enable the water-soluble conjugated polymer contrast agent to have the advantages of improving the signal-to-noise ratio and the imaging definition when being used for fluorescent imaging.
The invention also provides a preparation method of the water-soluble conjugated polymer contrast agent with the near infrared second window for absorbing fluorescence, which comprises the following steps:
s1, preparing a polymer I with a chemical structure shown in the following formula I:
wherein x=y=4 to 8.
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 the following formula II:
wherein m=30 to 50;
s3, removing succinimide ester from the polymer 2, and carrying out amidation reaction with methoxy polyethylene glycol amino to obtain a polymer 3, namely the water-soluble conjugated polymer contrast agent, wherein the structural formula of the water-soluble conjugated polymer contrast agent is shown as a formula III.
According to the preparation method of the water-soluble conjugated polymer contrast agent, the polymer 1 with the structure shown in the formula I with a donor-acceptor structure is used as a main chain, and is activated in an atom transfer radical polymerization mode to realize amidation reaction of amino groups, so that self-assembled nano particles with a large amount of PEG at the tail end of a side chain are synthesized. The degree of polymerization of the conjugated polymer 1 with the structure shown in the formula I is regulated, and the dosage of methoxy polyethylene glycol amino protected by the terminal amino is regulated to control the degree of polymerization of grafting, so that the regulation of properties such as molecular structure, nano size and the like is realized, and the regulation of targeting is realized.
For example, in the structure shown in formula II, the end capping group attached to the thiophene ring isThe end-capping group attached to the benzene ring is a halo group, such as bromo.
Preferably, in the step S1, the polymer I is prepared by carrying out a steller coupling reaction on a monomer 1 having a structural formula shown in formula iv, a monomer 2 having a structural formula shown in formula V, and a monomer 3 having a structural formula shown in formula vi, and the synthetic route is as follows:
specifically, monomer 1 is: 4, 7-dibromobenzo [1,2-c:4,5-c' ] bis ([ 1,2,5] thiadiazole, structural formula IV;
monomer 2 is: 2, 6-bis (trimethyltin) -4, 8-bis (5- (2-ethylhexyl) thiophen-2-yl) -benzodithiophene having formula V;
monomer 3 is: (2, 7-dibromo-9H-fluorene-9, 9-diyl) bis (propane-3, 1-diyl) bis (2-bromo-2-methylpropionate) having the structural formula VI.
Preferably, the step S1 specifically includes: 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 to perform deoxidization treatment, adding a palladium catalyst into the solution, reacting for 2-4 hours at 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. Among them, the palladium catalyst is preferably bis (triphenylphosphine) palladium (II) dichloride, and the solvent is preferably toluene.
Preferably, in the step S1, monomer 1: monomer 2: the molar ratio of the monomer 3 to the feed is 1:2:1.
preferably, the step S1 specifically includes: under a dark environment, 0.2mM 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 polymer 1 and acrylic acid-N-succinimidyl ester into an organic solvent under the condition of avoiding light, then adding a catalyst, adding ligand 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 polymer 2; the synthetic route is as follows:
preferably, the molar ratio of polymer I to N-succinimidyl acrylate is between 1:50 and 200, for example between 1:100.
Preferably, the catalyst is cuprous bromide and the organic solvent is anisole.
Preferably, the step S2 specifically includes:
100mg of polymer 1, 887mg of acrylic acid-N-succinimidyl ester are added into 1mL of anisole under the dark condition, 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 the mixture is repeatedly settled 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 methoxy polyethylene glycol amino into DMF (N, N-dimethylformamide) for dissolution, adding triethylamine (phase transfer catalyst) under the protection of nitrogen, and stirring at 35-45 ℃ for reaction for 24-60h; 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; the synthetic route is as follows:
preferably, the step S3 specifically includes:
under the condition of avoiding light, 300mg of polymer 2 and 630mg of methoxy polyethylene glycol amino are added into 5mL of DMF for dissolution, 177mg of triethylamine is added under the protection of nitrogen, and stirring reaction is carried out for 48h at 40 ℃; and 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 the molecular weight cut-off of 3500, dialyzing with deionized water for 48h, changing the dialyzate for 1 time every 6h, and freeze-drying the solution in the dialysis bag after the dialysis is finished to obtain the polymer 3.
The invention also provides application of the water-soluble conjugated polymer contrast agent in near-infrared second window fluorescence imaging.
The beneficial effects of the invention are as follows:
the water-soluble conjugated polymer contrast agent provided by the invention 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 conjugated bond length in the conjugated skeleton can be prolonged, the absorption is red-shifted, the fluorescence intensity of the polymer is improved, and the living body contrast resolution ratio and contrast ratio are improved; the PEG molecules with amino groups are selected as side chains, and are connected through covalent bonds, so that the near infrared two-region conjugated polymer has good water solubility, and tumor enrichment is realized through Enhancing Permeability and Retention (EPR) effect, so that the effect of fluorescence imaging is realized. The above-mentioned properties of the polymer make it advantageous to improve the signal-to-noise ratio and imaging definition when used in fluorescence imaging;
the water-soluble conjugated polymer contrast agent provided by the invention has good water solubility and higher near infrared two-region fluorescence imaging intensity, and can obviously improve the near infrared two-region fluorescence imaging effect.
Drawings
FIG. 1 is an absorption spectrum of a polymer 1 used in example 1;
FIG. 2 is an absorption spectrum of the polymer 2 used in example 1;
FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of the water-soluble conjugated polymer contrast agent (Polymer 3) prepared in example 1;
FIG. 4 is a transmission electron micrograph of the water-soluble conjugated polymer contrast agent prepared in example 1;
FIG. 5 is a schematic diagram showing the nano-diameter measured by dynamic light scattering of the water-soluble conjugated polymer contrast agent prepared in example 1;
FIG. 6 is a graph showing absorption and emission spectra of the water-soluble conjugated polymer contrast agent (2 mg/mL) prepared in example 1;
FIG. 7 is a near infrared and fluorescence imaging chart of a second window of the aqueous solution of the water-soluble conjugated polymer contrast agent prepared in example 1;
FIG. 8 shows the result of the biotoxicity test of the water-soluble conjugated polymer prepared in example 1.
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 preparation method of a water-soluble conjugated polymer contrast agent with near infrared second window for absorbing fluorescence comprises the following steps:
s1, preparing a polymer I with a chemical structure shown in the following formula I:
wherein x=y=4 to 8.
The method comprises the following specific steps:
in a dark environment, 4, 7-dibromobenzo [1,2-c:4,5-c' ] bis ([ 1,2,5] thiadiazole (35.2 mg,0.2 mM), donor molecule (monomer 2) 2, 6-bis (trimethyltin) -4, 8-bis (5- (2-ethylhexyl) thiophen-2-yl) -benzodithiophene (180.9 mg,0.2 mM), donor molecule (monomer 3) (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 the reaction tube, after dissolving the above 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 reacted under nitrogen protection at 80℃for 3 hours, and the crude polymer was filtered with a filter membrane and the brown polymer was removed by repeating the filtration to obtain a flask (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 the following formula II:
wherein m=30 to 50.
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 repeatedly settled in diethyl ether to obtain a reddish-brown acrylic acid-N-succinimidyl ester side chain-substituted conjugated main chain polymer, polymer 2 (900 mg).
S3, removing succinimide ester from the polymer 2, and carrying out amidation reaction with methoxy polyethylene glycol amino to obtain a polymer 3, namely the water-soluble conjugated polymer contrast agent, wherein the structural formula of the water-soluble conjugated polymer contrast agent is shown as a formula III.
The method comprises the following specific steps:
polymer 2 (300 mg), methoxypolyethylene glycol amino (630 mg,0.63 mmol) was added to the flask under dark conditions, and the mixture was dissolved in 5mL of DMF. Triethylamine (177 mg,1.75 mmol) was added under nitrogen protection and reacted at 40 ℃ for 48h with stirring; and (3) dropwise adding the solution in the product system into 20mL of deionized water after the reaction is finished, placing the obtained mixed aqueous solution into a dialysis bag (the molecular weight cut-off is 3500), dialyzing with deionized water for 48h, changing the dialyzate for 1 time every 6h, thoroughly removing DMF to obtain the aqueous solution of the polymer 3, and freeze-drying the aqueous solution of the content in the dialysis bag after the dialysis is finished to remove water, 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 was prepared by using methylene dichloride as an organic solvent, and the absorption spectrum was tested, and as shown in fig. 1, the absorption peak was found to be located in 734 nm band.
2. The organic solution of the polymer 2 was prepared by using methylene dichloride as an organic solvent, and the absorption spectrum was tested, and as shown in FIG. 2, the absorption peak was found to be located in the 727 nm band.
3. Polymer 3 prepared in example 1 was subjected to nuclear magnetic resonance spectroscopy to obtain a polymer 3 nuclear magnetic resonance hydrogen spectrum, and as shown in FIG. 3, the characteristic proton signal of the PEG side chain was seen.
4. Polymer 3 prepared in example 1 was formulated as an aqueous solution at a concentration of 2 mg/mL:
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 is apparent from FIG. 4 that the particle size of the polymer III was about 100 nm.
ii) based on the formulated aqueous polymer 3 solution, measured according to dynamic light scattering: the average hydrodynamic diameter of the water-soluble near infrared two-region fluorescence imaging in water is about 90 nanometers, and the resulting hydrodynamic diagram is shown in fig. 5.
iii) Based on the formulated aqueous solution of polymer 3, the absorption and emission spectra of polymer III were separately tested, and the results are shown in fig. 6, as can be seen: the absorption peak of the polymer III is positioned in 980 nm wave band, and the emission peak of the polymer III is positioned in 1050 nm wave band under 808nm laser excitation.
5. Polymer 3 prepared in example 1 was prepared as an aqueous solution of Polymer 3 having a concentration gradient of 5mg/mL, 3mg/mL, 2mg/mL, 1mg/mL, respectively.
Based on the prepared aqueous solutions of polymer 3 with different concentrations, the solution was excited by a 1064nm laser, and a fluorescence imaging chart of the solution was taken in a second near infrared window fluorescence imager, and the result is shown in fig. 7, wherein polymer III has a distinct fluorescence signal, which indicates that it has near infrared two-region fluorescence imaging performance.
6. The polymer 3 prepared in example 1 was prepared into a series of cell culture solutions having a concentration gradient, wherein the highest concentration of the polymer 3 in the cell culture solution was 3mg/mL, and these culture solutions were used to culture with mouse breast cancer cells for 4 hours, CCK-8 reagent was added, absorbance at 450 nm was measured, and the obtained absorbance was converted into cell viability, as shown in FIG. 8, and it can be seen that: the cytotoxicity of the polymer III is very low, which proves that the 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. The water-soluble conjugated polymer contrast agent is characterized by having a chemical structure shown in the following formula III:
wherein x=y=4 to 8, and m=30 to 50.
2. A method of preparing the water-soluble conjugated polymer contrast agent according to claim 1, comprising the steps of:
s1, preparing a polymer 1 with a chemical structure shown in the following formula I:
wherein x=y=4 to 8.
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 the following formula II:
wherein m=30 to 50;
s3, removing succinimide ester from the polymer 2, and carrying out amidation reaction with methoxy polyethylene glycol amino to obtain a polymer 3, namely the water-soluble conjugated polymer contrast agent, wherein the structural formula of the water-soluble conjugated polymer contrast agent is shown as a formula III.
3. The method for preparing a water-soluble conjugated polymer contrast agent according to claim 2, wherein in the step S1, the polymer I is prepared by carrying out a steller coupling reaction on a monomer 1 having a structural formula shown in formula iv, a monomer 2 having a structural formula V, and a monomer 3 having a structural formula vi, and the synthetic route is as follows:
4. the method for preparing a 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 of preparing a water-soluble conjugated polymer contrast agent according to claim 4, wherein in the step S1, monomer 1: monomer 2: the molar ratio of the monomer 3 to the feed is 1:2:1.
6. the method for preparing a water-soluble conjugated polymer contrast agent according to claim 5, wherein the step S1 specifically comprises: under a dark environment, 0.2mM 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 water-soluble conjugated polymer contrast agent according to claim 3, 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 water-soluble conjugated polymer contrast agent according to claim 7, wherein the molar ratio of the polymer I to the acrylic acid-N-succinimidyl ester is 1:50-200.
9. The method for preparing a water-soluble conjugated polymer contrast agent according to claim 8, wherein the step S2 specifically comprises:
100mg of polymer 1, 887mg of acrylic acid-N-succinimidyl ester are added into 1mL of anisole under the dark condition, 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 the mixture is repeatedly settled in diethyl ether, so that polymer 2 is obtained.
10. The method for preparing a 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 methoxy polyethylene glycol amino into DMF for dissolution, adding triethylamine under the protection of nitrogen, and stirring at 35-45 ℃ for reaction for 24-60h; 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; the synthetic route is as follows:
11. the method for preparing a 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 630mg of methoxy polyethylene glycol amino are added into 5mL of DMF for dissolution, 177mg of triethylamine is added under the protection of nitrogen, and stirring reaction is carried out for 48h at 40 ℃; and 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 the molecular weight cut-off of 3500, dialyzing with deionized water for 48h, changing the dialyzate for 1 time every 6h, and freeze-drying the solution in the dialysis bag after the dialysis is finished to obtain the polymer 3.
12. Use of a water-soluble conjugated polymer contrast agent as claimed in claim or prepared by a method as claimed in any one of claims 2 to 11 in near infrared second window fluorescence imaging.
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