CN110368503B

CN110368503B - PEG (polyethylene glycol) core cross-linked star-shaped high-molecular nano contrast agent as well as preparation method and application thereof

Info

Publication number: CN110368503B
Application number: CN201810329542.1A
Authority: CN
Inventors: 于靖; 赵鹏; 蔡雯婷; 陈起静; 范佳淇
Original assignee: Shanghai Tenth Peoples Hospital
Current assignee: Shanghai Tenth Peoples Hospital
Priority date: 2018-04-13
Filing date: 2018-04-13
Publication date: 2021-10-29
Anticipated expiration: 2038-04-13
Also published as: CN110368503A

Abstract

The invention relates to a PEG (polyethylene glycol) core cross-linked star-shaped polymer nano contrast agent which is prepared from star-shaped polymers taking PEG as a core through bonding fluorescent groups. The invention also relates to the application of the PEG nuclear cross-linked star-shaped macromolecular nano-contrast material in the preparation of fundus angiographic agents. Currently, fundus angiography clinically applied, such as fluorescein sodium or indocyanine green, causes adverse reactions when part of patients are injected with contrast agents intravenously, and the patients cannot complete contrast examination. The S-PPEGMA of the present invention is superior to the existing contrast agents₁₀ICG has the advantages of lower injection concentration and clearer choroidal development, and can reduce the side effect of the original medicament by reducing the administration concentration. Furthermore, S-PPEGMA₁₀Successful construction of ICG also provides an alternative to patients with allergies or other adverse reactions to the injection of existing contrast agents, enabling the patient to be diagnosed and treated in a timely manner.

Description

PEG (polyethylene glycol) core cross-linked star-shaped high-molecular nano contrast agent as well as preparation method and application thereof

Technical Field

The invention relates to the technical field of fundus angiography, in particular to a PEG nuclear cross-linked star-shaped macromolecular nano contrast agent and a preparation method and application thereof.

Background

Age-related macular degeneration (AMD) is one of the leading causes of central vision loss and irreversible blindness in elderly people over the age of 50, with about 2100 million patients with AMD currently occurring worldwide. AMD is classified into dry and wet types according to the presence or absence of new blood vessels. Early dry AMD, manifested as drusen, does not affect vision seriously, progresses to the late stage, and causes geographic atrophy, discoid degeneration and the like, resulting in decreased vision. In wet AMD, the macula is bleeding, edematous and severely impaired vision due to the appearance of Choroidal Neovascularization (CNV) in the macula. Currently, diagnostic methods for AMD include: fundus photography, OCT, fundus angiography techniques, of which fundus angiography techniques play an important role in the diagnosis of AMD disease and observation of therapeutic effects. Typical AMD is easy to diagnose clinically, but early subretinal neovascularization and serous separation are difficult to find, and the fundus angiography technology can accurately and clearly display the position, size and shape of the subretinal neovascularization.

Fundus fluorescence angiography technology is an important method for diagnosing fundus diseases and evaluating curative effects, and fundus fluorescence angiography agents commonly used in clinic mainly comprise sodium fluorescein and indocyanine green (ICG). The contrast agent enters the circulatory system through the elbow vein, the laser excites the contrast agent to develop the eyeground blood vessels, and the damaged parts of the retinal blood vessels display abnormal fluorescence, so that the method can be used for diagnosing related eyeground diseases such as CNV. Although contrast agents are widely used clinically at present, some patients still have adverse reactions, such as nausea, vomiting, rash, and even anaphylactic shock and death. These patients cannot be examined with a contrast medium, and cannot be diagnosed unambiguously for subsequent treatment. Even if a radiographic examination can be made, an appointment is generally required, and the patient requires at least one month for a definitive diagnosis. This results in a prolonged treatment period for the patient, delaying the timing of the treatment. Therefore, it is necessary to synthesize a contrast agent with low adverse reactions. Polyethylene glycol (PEG) is a water-soluble polyether polymerized from ethylene oxide and water or ethylene glycol. PEG is soluble in water and most organic solvents, has the characteristics of no toxicity, low immunogenicity and the like, and can be discharged out of the body through the kidney. The star-shaped polymer is a topological structure with a plurality of linear arms radiating outwards from a central core. The whole body is in a high-density spherical shape, the unique configuration of the high-density spherical shape can provide larger steric hindrance, the flexible arm can provide more functional sites, and the chelating group realizes targeting and fluorescent labeling. The PEG polymer has the characteristics of nano-scale size, colloid stability, low cytotoxicity, intracellular gene controlled release and pH response, and can stably play a carrying role. Therefore, PEG is a safe and effective transfer carrier.

Chinese patent 201210547672.5 discloses the use of FITC-dextran in the preparation of fundus angiographic agents, and more particularly discloses the use of a polymer linked with fluorescein derivatives in the preparation of angiographic agents, wherein the polymer linked with fluorescein derivatives is fluorescein isothiocyanate-dextran, NHS-fluorescein dextran, pentafluorophenyl ester dextran, Alexa 488 dextran, FluoProbes 488 dextran, DyLight 488 dextran, fluorescein isothiocyanate d-glucose polymer, fluorescein isothiocyanate chitin. In the prior art, the PEG nuclear crosslinking star-shaped macromolecular nano contrast agent for fundus angiography is not reported at present.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a PEG (polyethylene glycol) nuclear cross-linked star-shaped macromolecular nano contrast agent for fundus angiography.

The second purpose of the invention is to provide the application of the PEG nuclear cross-linked star-shaped macromolecular nano contrast agent in the preparation of fundus angiographic contrast agents aiming at the defects in the prior art.

In order to achieve the first purpose, the invention adopts the technical scheme that:

a PEG nuclear cross-linked star-shaped polymer nano contrast agent for fundus angiography is prepared from star-shaped polymers with PEG as a core through bonding fluorescent groups.

In a preferred embodiment of the present invention, the PEG-core star-shaped polymer is S-PPEGMA₁₀A star-shaped polymer.

As a preferred embodiment of the present invention, the fluorescent group is indocyanine green.

As a preferred embodiment of the present invention, said S-PPEGMA₁₀The star-shaped polymer is prepared by the following steps: using L-PPEGMA₁₀Linear arm, mixed solution in water and ethanolIn the formulation, it is prepared by RAFT dispersion polymerization.

As a preferred embodiment of the present invention, said L-PPEGMA₁₀The linear arm preparation method is as follows: monomeric PEGMA is polymerized in 1, 4-dioxane using small molecule CTA.

As a preferred embodiment of the present invention, the molecular weight of the monomeric PEGMA is 200-500, and more preferably, the molecular weight of the monomeric PEGMA is 300.

As a preferred embodiment of the present invention, the method for bonding indocyanine green is as follows: mixing S-PPEGMA₁₀Dissolving star-shaped macromolecule in DMSO, adding NHS and DCC, stirring, adding indocyanine green and TEA, reacting, dialyzing, and filtering.

As a preferred embodiment of the present invention, the method for bonding indocyanine green is as follows: collecting 0.5g S-PPEGMA₁₀Dissolving star polymer in DMSO, adding 33.8mg NHS and 60.5mg DCC, stirring for 5min, adding 1mg ICG and 10 μ L TEA, stirring for reaction, dialyzing in water phase, filtering, and freeze drying.

As a preferred embodiment of the present invention, said L-PPEGMA₁₀The linear arm preparation method is as follows:

small molecule CTA (0.925g,2.22mmol), PEGMA (10g,33mmol) and internal reference DMF (0.329g,4.5mmol) were dissolved in 22mL dioxane at 0 deg.C N₂And (3) after the oxygen is removed by bubbling for 30min, placing the mixture in an oil bath pan at 70 ℃ for heating. After the temperature had stabilized, a dioxane solution of AIBN (33.7mg,0.2mmol) after the oxygen removal was rapidly injected with a syringe. And (3) carrying out polymerization reaction for 6h, then dropwise adding the solution into n-hexane, precipitating the polymer, centrifugally collecting the polymer in the solution, dissolving the polymer with THF, and then dropwise adding the polymer into the n-hexane again for secondary precipitation and purification.

As a preferred embodiment of the present invention, said S-PPEGMA₁₀The star-shaped polymer is prepared by the following steps:

mixing L-PPEGMA₁₀maroCTA (0.4g,0.117mmol) was dissolved in 100mL of a mixed solvent of water and ethanol at a volume ratio of 80/20, and then BAC (0.188g,0.7mmol) as a crosslinking agent was added. Placing the mixture in an ice-water bath N₂Bubbling oxygen removalAfter 30min, the mixture is placed in an oil bath pan at 70 ℃ for heating. After the temperature was constant, an aqueous solution of initiator V-50 (6.6mg,0.024mmol) was quickly poured into the reaction flask. The polymerization was carried out for 1h, followed by dialysis (MWCO 30kg/mol) in an aqueous phase to remove unpolymerized polymer arms and finally freeze-drying to give star polymers.

In order to achieve the second object, the invention adopts the technical scheme that:

the PEG nuclear cross-linked star-shaped macromolecular nano contrast agent is applied to the preparation of fundus angiographic agents.

Currently, fundus angiography clinically applied, such as fluorescein sodium or indocyanine green, causes adverse reactions, such as nausea, vomiting, rash and even anaphylactic shock and death, when part of patients are injected with contrast agents intravenously. These patients cannot be examined with a contrast medium, and cannot be diagnosed unambiguously for subsequent treatment. Compared with the existing contrast agent, the PEG nuclear cross-linked star-shaped macromolecular nano contrast agent S-PPEGMA of the invention₁₀ICG has the advantage of lower injection concentration and clearer choroidal visualization, thus reducing the side effects of the original drug by reducing the concentration administered. Furthermore, S-PPEGMA₁₀Successful construction of ICG also provides an alternative to patients with allergies or other adverse reactions to the injection of existing contrast agents, enabling the patient to be diagnosed and treated in a timely manner.

The invention introduces the nano material into the retina angiography for the first time: by utilizing the structure and performance advantages of the nano material, the toxicity or sensitization of the traditional contrast agent is reduced while the contrast effect is maintained;

drawings

FIG. 1A is an NMR characterization of the solution before and after polymerization, with CDCl as solvent₃。

FIG. 1B shows L-PPEGMA₁₀The mobile phase is DMF.

FIG. 2A shows S-PPEGMA₁₀The mobile phase is DMF.

FIG. 2B shows S-PPEGMA₁₀Hydrodynamic diameter of (a).

FIG. 3A is a standard curve of ICG UV-VIS absorption spectrum.

FIG. 3B shows S-PPEGMA₁₀-uv absorption peak of ICG.

FIG. 4A shows 0-400nmol/l S-PPEGMA₁₀Effect of ICG on ARPE-19 and HUVEC cell Activity.

FIG. 4B shows 0, 100, 200, 400nmol/l S-PPEGMA₁₀ICG on ARPE-19 and HUVEC cells 24h after cell morphology change.

FIG. 4C shows 0-400nmol/l S-PPEGMA₁₀-effect of ICG on ARPE-19 apoptosis.

FIG. 5 shows S-PPEGMA₁₀-weight gain tendency in rats after ICG tail vein injection (a); (B) the mass of each organ is increasing.

FIG. 6 shows the tail vein injection of S-PPEGMA₁₀Structural changes in the organs (heart, liver, spleen, kidney, brain, muscle) on

days

1, 3, 5, 7, and 14 after ICG.

Fig. 7 shows the retinal structure and ONL layer thickness variation. (A)0 day; (B)1 day; (C)3 days; (D)5 days; (E)7 days; (F) and 14 days.

FIG. 8 shows retinal function changes, tail vein injection of S-PPEGMA₁₀Days 1, 3, 5, 7, 14 after ICG.

Fig. 9 is a fundus angiography result. (A) ICG; (B) S-PPEGMA₁₀-ICG。

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.

Example 1 preparation of PEG core-crosslinked Star-shaped Polymer Nanograph contrast agent

1. Material

Polyethylene glycol methyl ether methacrylate (PPEGMA, M)_n300), Sigma-Aldrich, cat No.: 447935, respectively; 2-cyano-2-propyldodecyl trithiocarbonate (CTA), Sigma-Aldrich, Cat No.: 723037, respectively; 1, 4-dioxane, Chinese medicine, product number: 10008918, respectively; 2,2' -Azobis (2-methylpropionitrile) (AIBN, CP), alatin, cat #: a104256; 2, 2' -azobis (2-methylpropionamidine) dihydrochloride (V-50, 97%), Sigma-Aldrich, cat #: 440914, respectively; n, N' -bis (acryloyl) cystamine (BAC, 98%), Alfa Aesar, cat #: a-4929. BN rat purchased from Beijing Wittiulihua laboratory animal technology Co., Ltd; ICR mice were purchased from shanghai slek laboratory animals, llc; human retinal pigment epithelial cell ARPE-19 cell line was purchased from iCell Bioscience; human umbilical vein endothelial cells were purchased from scienell; ICG amine was purchased from AAT Bioquest, cat #: 188; cell viability assay kit (CCK-8) was purchased from YEASEN corporation, cat #: 40203ES 60; proparacaine hydrochloride was purchased from Alcon; the compound tropicamide is purchased from Shentian for pharmacy; other reagents are all domestic analytical purifiers.

2. Method of producing a composite material

2.1 Synthesis of L-PPEGMA₁₀Linear arm

L-PPEGMA₁₀Linear arm, also a macromolecular chain transfer agent (MacroCTA), is prepared by polymerizing the monomer PEGMA (M) in 1, 4-dioxane using a small molecule of CTA by the RAFT process_n300) was prepared. The specific synthesis steps are as follows: the solids content of the monomer was 50% w/v, the molar ratio of CTA: PEGMA: AIBN was 1: 15: 0.1. small molecule CTA (0.925g,2.22mmol), PEGMA (10g,33mmol) and internal reference DMF (0.329g,4.5mmol) were dissolved in 22mL dioxane and then N at 0 deg.C₂After the oxygen is removed by bubbling for 30min, the mixture is placed in an oil bath pan at 70 ℃ for heating. After the temperature had stabilized, a dioxane solution of AIBN (33.7mg,0.2mmol) after the oxygen removal was rapidly injected with a syringe. The polymerization was carried out for 6h, and the monomer conversion was determined to be 68% by nuclear magnetism. And then dropwise adding the solution into n-hexane to precipitate the polymer, centrifugally collecting the polymer in the solution, dissolving the polymer by using THF, and then dropwise adding the dissolved polymer into the n-hexane again to perform secondary precipitation purification. The product was rotary evaporated and dried in vacuo to give 7.01g of a yellow viscous liquid in 65% yield. M_n,th＝3400g mol^-1,M_n＝4400g mol^-1(RI-GPC),

¹H NMR(500MHz,CDCl₃):4.04ppm(s,–COOCH₂–),3.78–3.49ppm(m,–O(CH₂)₂O–),3.32ppm(s,–OCH₃),1.98–1.66ppm(m,backbone–CH₂–),1.05–0.72ppm(s,–CH₃).

2.2 Synthesis of S-PPEGMA₁₀Star-shaped polymer

S-PPEGMA₁₀The star-shaped polymer is prepared by RAFT dispersion polymerization in a mixed solvent of water and ethanol. The preparation process comprises the following steps: mixing L-PPEGMA₁₀maroCTA (0.4g,0.117mmol) was dissolved in 100mL of a mixed solvent of water and ethanol at a volume ratio of 80/20, and then BAC (0.188g,0.7mmol) as a crosslinking agent was added. Placing the mixture in an ice-water bath N₂Deaerating by bubbling for 30min, and heating in an oil bath pan at 70 ℃. After the temperature was constant, an aqueous solution of initiator V-50 (6.6mg,0.024mmol) was quickly poured into the reaction flask. The polymerization was carried out for 1h, followed by dialysis (MWCO 30kg/mol) in aqueous phase to remove unpolymerized polymer arms and finally freeze-drying to give star-shaped polymers in 94% yield. RI-GPC: M_n＝56.2kg mol^-1,

2.3 Star-shaped Polymer Supported Indocyanine Green ICG (S-PPEGMA)₁₀Preparation and characterization of-ICG)

Weighing 0.5g S-PPEGMA₁₀The star polymer was dissolved in DMSO, and then 33.8mg of NHS and 60.5mg of DCC were added, and after stirring for 5min, 1mg of ICG and 10. mu.L of TEA were added, and the reaction was stirred for 24 hours. Then dialyzing in water phase (MWCO 30kg/mol) to obtain blue clear transparent solution, filtering, and freeze-drying to obtain flocculent material.

3.S-PPEGMA₁₀Synthesis and characterization of ICG

3.1 Synthesis of L-PPEGMA₁₀Linear arm and characterization results

Preparation of L-PPEGMA by RAFT homogeneous polymerization₁₀Linear arm, L-PPEGMA₁₀The test characterization of (2) is shown in fig. 1. FIG. 1A NMR characterization of the solution before and after polymerization, with CDCl as solvent₃(ii) a FIG. 1B L-PPEGMA₁₀The mobile phase is DMF.

3.2 Synthesis of S-PPEGMA₁₀Star-shaped polymer

Preparation of S-PPEGMA by RAFT heterogeneous polymerization₁₀Star-shaped polymer, FIG. 2A is S-PPEGMA₁₀The mobile phase is DMF; FIG. 2B shows S-PPEGMA₁₀Hydrodynamic diameter of (a).

3.3S-PPEGMA₁₀Test characterization of ICG

First, the ultraviolet-visible absorption spectrum of ICG with different concentrations is tested, the highest absorption peak is 795nm, and a calibration curve is drawn as shown in FIG. 3 (A). The S-PPEGMA was then tested₁₀The absorption intensity at 795nm of ICG at a solubility of 6mg/mL was 1.51487 (FIG. 3B), which was finally calculated to give two star-shaped macromolecules loaded with one ICG.

Example 2S-PPEGMA₁₀Verification of the safety and effectiveness of ICG at the cellular and animal level

1. Materials, methods

1.1CCK8 experiment

Taking ARPE-19 and HUVEC cells in logarithmic growth phase, paving the cells into a 96-well plate at 5000/hole, putting the 96-well plate into an incubator for culturing for 24h, and waiting for the cells to adhere to the wall. Respectively adding 0-400nmol/l ICG and S-PPEGMA₁₀ICG, incubation for 24 h. And (4) changing the solution, adding 110uL of culture medium containing 10uL of CCK-8 into each well, and incubating for 2h at 37 ℃ in a dark incubator. And (4) detecting on a computer, selecting the wavelength of 450nm, and detecting the OD value of each hole on a multifunctional microplate reader.

1.2 flow cytometry

Taking ARPE-19 in logarithmic phase, spreading the ARPE-19 in a 12-hole plate at 5 ten thousand/hole, putting the ARPE-19 in an incubator for culturing for 24 hours until the cells adhere to the wall. Adding 0, 40, 80, 100, 200, 400nmol/l ICG and S-PPEGMA respectively₁₀ICG, incubation for 24 h. Removing culture medium by suction, washing with 1xPBS for 2 times, adding 1 Xpancreatin for 2 min, adding culture medium to stop digestion, and collectingThe cells of each group were centrifuged at 1000rmp for 5min, the supernatant was discarded, washed with PBS for 2 times, centrifuged again, the supernatant discarded, 50ul of 1 XBinding Buffer and 2.5ul of APC Annexin V were added to each tube, and protected from light at room temperature for 15min, then 250ul of 1 XBinding Buffer and 5ul of PI were added, and protected from light at room temperature for 5 min. And (4) detecting by an up-flow cytometer.

1.3 Paraffin section preparation

12 BN rats were divided into 2 groups, control group (tail vein injection of physiological saline, n-2), S-PPEGMA₁₀ICG group (tail vein injection 0.05mg/ml 0.2ml S-PPEGMA)₁₀-ICG, n ═ 10), the eyeball and the organs of the whole body (heart, liver, spleen, lung, kidney, brain, muscle) were harvested on

days

1, 3, 5, 7, 14 after tail vein injection, respectively, and fixed with 4% paraformaldehyde for 24 h. Taking out the fixed eyeballs, washing the fixed eyeballs with running water, and sequentially putting the eyeballs and the organs into low-concentration to high-concentration gradient ethanol at normal temperature for dehydration, wherein the process is as follows: 50%, 70%, 80%, 90%, 95% ethanol for 1h each; anhydrous ethanol for 30-45min, xylene for 30min, and then placing into xylene for transparency. Sequentially immersing the specimen in a xylene paraffin mixed solution (xylene: soft wax is 1: 1) for 10 min; melted pure paraffin (52-54 ℃) for 50min, (56-58%) for 1 h. And (4) embedding the waxed tissue in an embedding device, and preserving after the waxed tissue is completely condensed and hardened. The sections were cut with a paraffin microtome, and the eyeball was sectioned from front to back with a thickness of 5um, with the axis perpendicular to the line from the center of the cornea to the optic nerve root. Flattening the wax sheet in warm water, mounting on a glass slide, and drying in a constant temperature box at 37 ℃ for later use.

1.4HE staining

Placing the slices in an oven at 60 deg.C for 30min, and dewaxing the slices in xylene I, II for 10min respectively; the slices were sequentially passed through 100%, 95%, 85%, 75% ethanol down to deionized water hydration for 5min each. Staining with hematoxylin for 3 min; flushing with running water for 5 min; washing with flowing water for 5min after differentiation with 1% hydrochloric acid alcohol; staining with eosin for 1 min; and (3) performing ascending gradient ethanol dehydration, enabling dimethylbenzene to be transparent, sealing a piece with neutral gum, and observing and collecting an image under a microscope.

1.5 Electroretinogram (ERG)

The visual electrophysiological detection system is adopted to record F-ERG, the stimulator is a full-field stimulator, the recording electrode is a gold foil annular cornea electrode, the reference electrode and the grounding electrode are stainless steel needle-shaped electrodes respectively, and the impedance of each electrode is less than 5 omega. F-ERG recordings were carried out using a simultaneous binocular recording in rats. F-ERG records that the rat is dark adapted for 12h before, 1% sodium pentobarbital is injected in 40mg/kg abdominal cavity, 0.1ml of fast-sleep new injection is injected intramuscularly, proparacaine hydrochloride is locally anesthetized on the surfaces of the two eyes, electrodes are arranged under dark red light, the recording electrodes are respectively arranged on the cornea of the two eyes, the cornea is kept moist by local physiological saline, reference electrodes are respectively penetrated into the middle skin of the rat arch of eyebrows, grounding electrodes are arranged on the tail, electrophysiological software (Kanghuaruiming vision electrophysiological system) is opened, waveforms are stimulated and collected in a 9-channel mode, and the interval of each stimulation is 3 min.

1.6 fundus angiography technique

BN male rats (8 weeks, the mass is 180 +/-10 g) are taken and injected with 40mg/kg of 1% pentobarbital sodium in an abdominal cavity, compound tropicamide mydriasis is performed after anesthesia, and fundus photography under no red light and autofluorescence is respectively performed on two groups of rats. Injecting indocyanine green (ICG) with different concentration gradients and star-shaped polymer-loaded indocyanine green ICG (S-PPEGMA) into tail vein respectively₁₀-ICG), ICG and S-PPEGMA₁₀-ICG concentrations are: 0.2ml was injected at 0.02mg, 0.01mg and 0.001mg, and the change of the contrast was dynamically observed for 60 minutes.

2. Results

2.1 cellular level validation of S-PPEGMA₁₀Security of ICG

The results of CCK8 show that 0-400nmol/l S-PPEGMA₁₀ICG was not toxic in both ARPE-19 cells and HUVEC cell lines, and its safety was confirmed at the cellular level, with no statistical significance for the differences between groups (FIG. 4A); 0. 100, 200, 400nmol/l S-PPEGMA₁₀After ICG treatment of ARPE19 cells 24, the cells of each group mostly had a flat polygonal or cobblestone-like appearance with a small amount of pigment particles in the cells and no morphological changes in the treated groups; after HUVEC cells are treated by the same method, each group of cells present typical cobblestone-like arrangement, the boundary is clear, the morphological change is avoided, and the result shows that S-PPEGMA₁₀ICG had no effect on cell morphology (fig. 4B); flow cytometry results showed 0-400nmol/l S-PPEGMA₁₀ICG did not cause apoptosis, as evidenced at the cellular levelThe safety was confirmed, and the results are shown in FIG. 4C.

2.2 animal level verification of S-PPEGMA₁₀Security of ICG

BN rat tail vein injection of 0.01mg S-PPEGMA₁₀At 1, 3, 5, 7, and 14 days after ICG, there was no significant change in the body weight and the growth tendency of each organ in rats compared with the control group (fig. 5A-B); the structures of all organs (heart, liver, spleen, kidney, brain and muscle) are not obviously damaged (figure 6); the result of HE staining of the fundus indicates that S-PPEGMA₁₀No significant change in retinal ONL layer thickness after ICG injection, indicating no destruction of retinal structure (fig. 7); the ERG results showed no significant difference in the amplitude of the a-and b-waves in rats compared to the control group (fig. 8), indicating no impairment of retinal function.

2.3S-PPEGMA₁₀Effectiveness of ICG in horizontal fundus angiography in animals

The fundus angiography result shows that 0.02mg, 0.01mg, 0.001mg ICG and S-PPEGMA are injected into the tail vein respectively₁₀After ICG, at equivalent concentrations, S-PPEGMA₁₀The ICG group visualised retinal and choroidal vessels more clearly, whereas the ICG group visualised blurred or only retinal main vessels (fig. 9).

The invention successfully constructs star-shaped polymer-loaded indocyanine green ICG (S-PPEGMA)₁₀-ICG), validated at cellular and animal levels for S-PPEGMA₁₀The safety of ICG lays a foundation for the next application of the nano material. Currently, fundus angiography clinically applied, such as fluorescein sodium or indocyanine green, causes adverse reactions, such as nausea, vomiting, rash and even anaphylactic shock and death, when part of patients are injected with contrast agents intravenously. These patients cannot be examined with a contrast medium, and cannot be diagnosed unambiguously for subsequent treatment. Compared with the existing contrast agent, S-PPEGMA₁₀ICG has the advantage of lower injection concentration and clearer choroidal visualization, thus reducing the side effects of the original drug by reducing the concentration administered. Furthermore, S-PPEGMA₁₀Successful construction of ICG also provides an alternative to patients with allergies or other adverse reactions to the injection of existing contrast agents, enabling the patient to be diagnosed and treated in a timely manner.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims

1. The PEG nuclear cross-linked star-shaped polymer nano contrast agent for fundus angiography is characterized in that the contrast agent is prepared by taking star-shaped polymers with PEG as cores and carrying fluorescent groups through bonds, and the star-shaped polymers with PEG as cores refer to S-PPEGMA₁₀Star-shaped polymer, the fluorescent group is indocyanine green, and the S-PPEGMA₁₀The star-shaped polymer is prepared by the following steps: using L-PPEGMA₁₀Linear arms prepared by RAFT dispersion polymerization in a mixed solvent of water and ethanol, said L-PPEGMA₁₀The linear arm preparation method is as follows: polymerizing monomer PEGMA in 1, 4-dioxane by adopting micromolecular CTA, wherein the molecular weight of the monomer PEGMA is 200-500;

the method for bonding indocyanine green comprises the following steps: mixing S-PPEGMA₁₀Dissolving star-shaped macromolecule in DMSO, adding NHS and DCC, stirring, adding indocyanine green and TEA, reacting, dialyzing, and filtering.

2. The PEG core-crosslinked star-shaped polymer nano contrast agent according to claim 1, wherein the method for bonding indocyanine green is as follows: collecting 0.5g S-PPEGMA₁₀Dissolving star polymer in DMSO, adding 33.8mg NHS and 60.5mg DCC, stirring for 5min, adding 1mg ICG and 10 μ L TEA, stirring for reaction, dialyzing in water phase, filtering, and freeze drying.

3. The use of the PEG core cross-linked star-shaped polymer nano-contrast agent as claimed in any one of claims 1 to 2 in the preparation of fundus angiographic agents.