A new-born blood vessel targeting near-infrared II region fluorescent emission nano-hydrogel and preparation and application thereof

Abstract

The application discloses a kind of new blood vessel targeting near-infrared II region fluorescent emission nano hydrogel and its preparation and application.The preparation method of nano hydrogel provided in the application comprises the following steps: (1) organic small molecule containing organic amine and organic small molecule containing boric acid group are dissolved in acid-containing ethanol solution to obtain carbon dot precursor solution;(2) the carbon dot precursor solution of step (1) is obtained by solvothermal reaction;(3) the ethanol dispersion of carbon dot of step (2) is added to fucoidan solution, and ultrasonic is obtained to obtain crude product;(4) the crude product of step (3) is treated by centrifugation, dialysis and freeze-drying, and the nano hydrogel is obtained.The application uses fucoidan with new blood vessel targeting as raw material, and carbon dot as fluorescent emission and photodynamic therapy function body, by adding different content, the preparation of near-infrared II region fluorescent emission nano hydrogel can be realized and applied to abnormal new blood vessel imaging and corresponding photodynamic therapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a near-infrared II region fluorescence emitting nano-hydrogel with neovascular targeting and its preparation and application. BACKGROUND

[0002] Vascular dysfunction, including abnormalities in vascular structure, hemodynamics and molecular expression related to vascular function, is closely related to the occurrence and development of various diseases. For example, diabetic retinopathy is a series of ocular fundus lesions caused by retinal microvascular leakage and obstruction due to chronic progression of diabetes. For another example, impaired cerebral vascular network due to loss or significant reduction of blood supply can lead to cell death and impaired brain function. Tumor angiogenesis and its abnormal vascular structure not only participate in the development of tumor by providing oxygen and nutrients, but also have a profound impact on tumor microenvironment, helping to increase drug resistance and invasiveness. Therefore, monitoring vascular abnormalities, especially vascular structure and hemodynamic abnormalities, is of great significance for guiding the diagnosis and treatment of related diseases. Near-infrared II region (900-1800nm) fluorescence can achieve deeper tissue penetration, higher spatial resolution and signal-to-noise ratio compared with visible light and near-infrared I region fluorescence, and has broad application prospects in the fields of tissue imaging and optical therapy. Nano-hydrogel based on carbon dots has good biocompatibility and biodegradability, and integrates the photoluminescence properties of carbon dots and the optical therapy function, which can promote in vivo near-infrared fluorescence imaging and potential clinical practice.

[0003] As provided in Chinese Patent No. CN114652843B, a nano-particle for targeted treatment of ocular neovascularization and a preparation method thereof are provided. The nano-particle comprises a targeting precursor molecule and an ultra-small nano-particle. The targeting precursor molecule is formed by two 4,4'-dipyridyl-zinc (II) molecules and one neovascular targeting polypeptide connected by a benzene ring. The ultra-small nano-particle is a gold / platinum-indocyanine green nano-composite. Thus, an image-guided ocular fundus neovascularization treatment nano-drug is constructed. The synthesis process parameters of the nano-drug are complex and need to be strictly controlled, and gold and platinum are used as raw materials, which is high in cost. SUMMARY

[0004] In view of this, the present application provides a near-infrared II region fluorescence emitting nano-hydrogel with neovascular targeting and its preparation and application.

[0005] The preparation method of the nano-hydrogel provided by the present application comprises the following steps:

[0006] (1) dissolving an organic small molecule containing an organic amine and an organic small molecule containing a boronic acid group in an acid-containing ethanol solution to obtain a carbon dot precursor solution;

[0007] (2) subjecting the carbon dot precursor solution of step (1) to a solvothermal reaction to obtain carbon dots;

[0008] (3) adding the ethanol dispersion of carbon dots of step (2) into a fucoidan solution and ultrasonically treating to obtain a crude product;

[0009] (4) subjecting the crude product of step (3) to centrifugation, dialysis and freeze-drying treatment to obtain the nano-hydrogel.

[0010] Optionally, in step (1), the organic small molecule containing an organic amine is at least one selected from ethylenediamine, aniline, o-phenylenediamine, m-phenylenediamine or p-phenylenediamine; and the organic small molecule containing a boronic acid group is at least one selected from phenylboronic acid, 1,4-phenyldiboronic acid or 3-aminophenylboronic acid.

[0011] Optionally, in step (1), the concentration of the organic small molecule containing an organic amine in the carbon dot precursor solution is 1 g / L to 10 g / L; and the concentration of the organic small molecule containing a boronic acid group in the carbon dot precursor solution is 1 g / L to 10 g / L.

[0012] Optionally, in step (1), the molar ratio of the organic small molecule containing an organic amine to the organic small molecule containing a boronic acid group in the carbon dot precursor solution is 1:1 to 1:5.

[0013] Optionally, in step (2), the solvothermal reaction is performed in a hydrothermal kettle, the reaction temperature is 140°C to 200°C, and the reaction time is 6 h to 24 h.

[0014] Optionally, in step (3), the mass percentage concentration of the fucoidan is 0.5% to 3%, and the amount of carbon dots added is 0.5 g / L to 3 g / L.

[0015] Optionally, in step (3), the ethanol dispersion of carbon dots is added dropwise into the fucoidan solution.

[0016] Optionally, in step (4), the centrifugation is performed at a speed of 5,000 rpm to 8,000 rpm; the dialysis is performed for 7 days to 14 days, deionized water is used as the dialysis liquid, the dialysis liquid is replaced once a day, the freeze-drying is performed at a temperature of -20 to -80°C, and the drying time is 3 to 7 days.

[0017] The nano-hydrogel prepared by the method also falls within the protection scope of the present application.

[0018] The application of the nano-hydrogel in new blood vessel fluorescence imaging and photodynamic therapy also falls within the protection scope of the present application.

[0019] Beneficial effects:

[0020] The present application uses natural polysaccharide fucoidan as raw material to simultaneously prepare boronic acid group-containing carbon dots that can produce near-infrared II region fluorescence. The fucoidan and the boronic acid group-containing carbon dots are crosslinked by using boronic ester bond reaction to prepare nanohydrogel. Compared with the prior art, the nanohydrogel of the present application can enrich the nanofunctional carbon dots in the nanohydrogel to realize efficient near-infrared II region fluorescence image-guided photodynamic therapy. BRIEF DESCRIPTION OF DRAWINGS

[0021] For the purpose of illustration and not limitation, the present application will now be described according to the preferred embodiments of the present application, in particular with reference to the accompanying drawings, in which:

[0022] Figure 1 Transmission electron microscope photo of the nanohydrogel prepared for the present application embodiment 1.

[0023] Figure 2 Near-infrared fluorescence emission spectrum of the nanohydrogel prepared for the present application embodiment 1. DETAILED DESCRIPTION

[0024] The technical solutions of the present application will be further described in detail below in combination with specific embodiments. It should be understood that the following embodiments are only illustratively described and explained the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope of protection intended by the present application.

[0025] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available goods, or can be prepared by known methods, if not specifically mentioned or processing techniques.

[0026] The fucoidan used in the experiment was purchased from Merck Company, and other chemical reagents were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0027] Example 1, preparation of near-infrared II region fluorescence emission nanohydrogel with neovascular targeting property

[0028] The present embodiment provides a near-infrared II region fluorescence emission nanohydrogel with neovascular targeting property, and the preparation method thereof comprises the following steps:

[0029] (1) Preparation of carbon dot precursor solution: first prepare acid ethanol solution, and the concentration of hydrochloric acid in the obtained acid ethanol solution (hydrochloric acid is used for preparation) is 1 mol / L; then dissolve ethylenediamine and 2-carboxyphenylboronic acid in the acid ethanol solution to obtain the carbon dot precursor solution, wherein the concentration of ethylenediamine is 1 g / L, the concentration of 2-carboxyphenylboronic acid is 1 g / L, and the molar ratio of ethylenediamine to 2-carboxyphenylboronic acid is 1:1.

[0030] (2) Take 30 mL of the carbon dot precursor solution obtained in step (1) and place it in a hydrothermal kettle with a volume of 100 mL. React in the hydrothermal kettle for 6 h at a reaction temperature of 140°C. After the reaction, obtain a hydrothermal product, carbon dots.

[0031] (3) Dissolve 1 g of fucoidan in 99 g of sterile phosphate buffer to prepare a fucoidan solution with a mass percentage concentration of 1%. Disperse the carbon dots obtained in step (2) in ethanol, and add the ethanol dispersion of the carbon dots dropwise to the fucoidan solution while stirring at a stirring speed of 1200 rpm / min. The final concentration of the carbon dots is 1 g / L, and a crude product is obtained.

[0032] (4) Centrifuge the crude product obtained in step (3) at a speed of 5000 rpm for 10 min, dialyze for 7 days, use a dialysis bag (produced by United Carbon Corporation, USA) with a molecular weight cut-off of 1000, and freeze dry (Toshiba) at -20°C for 4 days to obtain a nanohydrogel.

[0033] Detect the nanohydrogel prepared in this example:

[0034] 1. Photodynamic therapy performance of the nanohydrogel

[0035] Disperse the nanohydrogel in a cell-specific culture medium for human umbilical vein endothelial cells (HUVEC, purchased from ATCC) (purchased from ScienCell) at a concentration of 100 mg / mL, and perform light irradiation on the HUVEC, with a light irradiation wavelength of 808 nm, a light irradiation time of 2 min, and a light irradiation intensity of 0.2 W / cm 2 Under light irradiation, the photodynamic process of the nanohydrogel generates singlet oxygen, thereby killing the HUVEC. This model is used to evaluate the killing effect of the nanohydrogel on the endothelial cells of new blood vessels. The survival rate of the cells is detected by CCK-8.

[0036] 2. Size and morphology of the nanohydrogel

[0037] The size and morphology of the nanohydrogel are obtained from a transmission electron microscope (FEI Tecnai F20, Thermo Fisher Scientific) photograph. The diameter of the nanohydrogel is 46±7 nm Figure 1 , and a certain amount of carbon dots are contained therein.

[0038] 3. Fluorescence performance of the nanohydrogel

[0039] The fluorescence performance of the carbon dots is obtained from a near-infrared fluorescence emission spectrometer (FLS1000, Edinburgh Instruments). The test light source is a laser, and the concentration is 10 mg / mL.

[0040] From the near-infrared fluorescence emission spectrum, it can be seen that the fluorescence emission intensity of the nano-hydrogel is the highest at 1260 nm under the excitation of 808 nm near-infrared light Figure 2 ), and this waveband is located in the NIR II region (1000-1700 nm), and the fluorescence emission is derived from the carbon dots in the nano-hydrogel.

[0041] Example 2, preparation of a nano-hydrogel with new blood vessel targeting near-infrared II region fluorescence emission

[0042] The specific method and steps for preparing the nano-hydrogel with new blood vessel targeting near-infrared II region fluorescence emission in this example are the same as those in Example 1, except that in step (3), the final concentration of carbon dots is 2 g / L.

[0043] Example 3, preparation of a nano-hydrogel with new blood vessel targeting near-infrared II region fluorescence emission

[0044] The specific method and steps for preparing the nano-hydrogel with new blood vessel targeting near-infrared II region fluorescence emission in this example are the same as those in Example 1, except that in step (3), the final concentration of carbon dots is 3 g / L.

[0045] Example 4, preparation of a nano-hydrogel with new blood vessel targeting near-infrared II region fluorescence emission

[0046] The specific method and steps for preparing the nano-hydrogel with new blood vessel targeting near-infrared II region fluorescence emission in this example are the same as those in Example 1, except that in step (3), the final concentration of carbon dots is 4 g / L.

[0047] Example 5, preparation of a nano-hydrogel with new blood vessel targeting near-infrared II region fluorescence emission

[0048] The specific method and steps for preparing the nano-hydrogel with new blood vessel targeting near-infrared II region fluorescence emission in this example are the same as those in Example 1, except that in step (3), the mass percentage concentration of fucoidan solution is adjusted to 2%.

[0049] Example 6, preparation of a nano-hydrogel with new blood vessel targeting near-infrared II region fluorescence emission

[0050] The specific method and steps for preparing the nano-hydrogel with new blood vessel targeting near-infrared II region fluorescence emission in this example are the same as those in Example 1, except that in step (3), the mass percentage concentration of fucoidan solution is adjusted to 3%.

[0051] The nano-hydrogels prepared in Examples 1-6 are detected.

[0052] (1) Diameter of nano-hydrogel: detection method is the same as in Example 1.

[0053] (2) Nanohydrogel fluorescence emission peak position: detection method same as Example 1.

[0054] (3) HUVEC survival rate (%) without light: detection method same as Example 1.

[0055] (4) HUVEC survival rate (%) under light: detection method same as Example 1.

[0056] The detection results are shown in Table 1.

[0057] Table 1

[0058]

[0059]

[0060] The nanohydrogel is crosslinked by borate ester bonds between the boronic acid groups on the surface of the carbon dots and the adjacent hydroxyl groups in the fucoidan molecular chain, as well as hydrogen bonds and van der Waals forces and other physical interactions. As can be seen from Table 1, with the increase of the concentration of carbon dots (Example 1, Example 2, Example 3 and Example 4) or the concentration of fucoidan (Example 1, Example 5 and Example 6), the diameter of the nanohydrogel shows a significant upward trend. The fluorescence emission peak position of the nanohydrogel is determined by the carbon dots, depending on the structure and chemical composition of the carbon dots, and the concentration change does not affect the fluorescence emission peak position. In addition, the photodynamic effect is generated by the carbon dots, and with the increase of the concentration of carbon dots, the photodynamic killing effect on cells is enhanced, and fucoidan does not produce significant toxicity to cell survival.

[0061] Example 7, preparation of nanohydrogel with new blood vessel targeting near-infrared II region fluorescence emission

[0062] The specific method and steps for preparing the nanohydrogel with new blood vessel targeting near-infrared II region fluorescence emission in this example are the same as those in Example 1, except that in step (2), the reaction temperature in the hydrothermal kettle is 160°C, and the reaction time is 6h.

[0063] Example 8, preparation of nanohydrogel with new blood vessel targeting near-infrared II region fluorescence emission

[0064] The specific method and steps for preparing the nanohydrogel with new blood vessel targeting near-infrared II region fluorescence emission in this example are the same as those in Example 1, except that in step (2), the reaction temperature in the hydrothermal kettle is 180°C, and the reaction time is 6h.

[0065] Example 9, preparation of nanohydrogel with new blood vessel targeting near-infrared II region fluorescence emission

[0066] The specific method and steps for preparing the near-infrared II region fluorescence emission nanohydrogel with neovascular targeting in this example are the same as those in Example 1, except that in step (2), the reaction temperature in the hydrothermal kettle is 200 ℃, and the reaction time is 6 h.

[0067] Example 10, Preparation of a near-infrared II region fluorescence emission nanohydrogel with neovascular targeting

[0068] The specific method and steps for preparing the near-infrared II region fluorescence emission nanohydrogel with neovascular targeting in this example are the same as those in Example 1, except that in step (2), the reaction temperature in the hydrothermal kettle is 200 ℃, and the reaction time is 12 h.

[0069] The carbon dots in the nanohydrogels prepared in Examples 7-10 were detected, and the detection method was as follows:

[0070] 1. Size and morphology of carbon dots

[0071] The size and morphology of the carbon dots were obtained by transmission electron microscopy (FEI Tecnai F20, Thermo Fisher Scientific).

[0072] 2. Fluorescence performance of carbon dots

[0073] The fluorescence performance of the carbon dots was obtained by a near-infrared fluorescence emission spectrometer (FLS1000, Edinburgh Instruments), and the test light source was a laser with a concentration of 1 mg / mL.

[0074] 3. Singlet oxygen quantum yield of carbon dots: The singlet oxygen quantum yield was detected by a chemical analysis method, using 1,3-diphenylbenzofuran as a singlet oxygen detection probe and methylene blue as a reference, and the detection results are shown in Table 2.

[0075] Table 2

[0076]

[0077] By adjusting the temperature and time of the solvothermal reaction conditions, the morphology and fluorescence emission characteristics of the carbon dots can be adjusted. Comparing Examples 1, 7, and 8, as the reaction temperature increases and the reaction time increases (Examples 9 and 10), the particle size of the carbon dots gradually increases, the quantum yield of the carbon dots increases, and the fluorescence emission peak has a certain degree of red shift. The nanohydrogel realizes near-infrared II region imaging and photodynamic processes through carbon dots, and therefore, as the quantum yield of the carbon dots increases and the fluorescence emission peak red shifts, the nanohydrogel will have higher neovascular killing effect and near-infrared fluorescence imaging effect.

[0078] Example 11, Performance detection of nanohydrogel

[0079] The specific method and steps are the same as those in Example 1, except that in step (4), the concentration of the nanohydrogel is 50 μg / mL.

[0080] Example 12, Performance detection of nanohydrogel

[0081] The specific method and steps are the same as those in Example 1, except that in step (4), the concentration of the nanohydrogel is 200 μg / mL.

[0082] Example 13, Performance detection of nanohydrogel

[0083] The specific method and steps are the same as those in Example 1, except that in step (4), the light irradiation time is 30 s.

[0084] Example 14, Performance detection of nanohydrogel

[0085] The specific method and steps are the same as those in Example 1, except that in step (4), the light irradiation time is 5 min.

[0086] Example 15, Performance detection of nanohydrogel

[0087] The specific method and steps are the same as those in Example 1, except that in step (4), the light irradiation intensity is 0.1 W / cm 2 .

[0088] Example 16, Performance detection of nanohydrogel

[0089] The specific method and steps are the same as those in Example 1, except that in step (4), the light irradiation intensity is 0.4 W / cm 2 .

[0090] Table 3

[0091]

[0092] Comparing Comparative Example 1 and Examples 11-16, the concentration of the nanohydrogel and the light irradiation parameters can produce different killing effects on HUVEC. With the increase of the concentration (Example 11, Example 1 and Example 12), the extension of the light irradiation time (Example 13, Example 1 and Example 14) or the increase of the light irradiation intensity (Example 15, Example 1 and Example 16), the survival rate of HUVEC shows a downward trend, indicating that the photodynamic cell killing effect is gradually enhanced.

[0093] Example 17, Preparation of near-infrared II region fluorescence emitting nanohydrogel with neovascular targeting property

[0094] The specific method and steps for preparing the near-infrared II region fluorescence emitting nanohydrogel with neovascular targeting property in this example are the same as those in Example 1, except that the ethylenediamine used is replaced by an equimolar concentration of aniline.

[0095] Example 18, Preparation of a nanohydrogel with neovasculature-targeting near-infrared II region fluorescent emission

[0096] The specific method and procedure for preparing a nanohydrogel with neovasculature-targeting near-infrared II region fluorescent emission in this example is the same as in Example 1, except that the ethylenediamine used is replaced with an equimolar concentration of o-phenylenediamine.

[0097] Example 19, Preparation of a nanohydrogel with neovasculature-targeting near-infrared II region fluorescent emission

[0098] The specific method and procedure for preparing a nanohydrogel with neovasculature-targeting near-infrared II region fluorescent emission in this example is the same as in Example 1, except that the ethylenediamine used is replaced with an equimolar concentration of m-phenylenediamine.

[0099] Example 20, Preparation of a nanohydrogel with neovasculature-targeting near-infrared II region fluorescent emission

[0100] The specific method and procedure for preparing a nanohydrogel with neovasculature-targeting near-infrared II region fluorescent emission in this example is the same as in Example 1, except that the ethylenediamine used is replaced with an equimolar concentration of p-phenylenediamine.

[0101] Example 21, Preparation of a nanohydrogel with neovasculature-targeting near-infrared II region fluorescent emission

[0102] The specific method and procedure for preparing a nanohydrogel with neovasculature-targeting near-infrared II region fluorescent emission in this example is the same as in Example 1, except that the 2-carboxyphenylboronic acid used is replaced with an equimolar concentration of 3-carboxyphenylboronic acid.

[0103] Example 22, Preparation of a nanohydrogel with neovasculature-targeting near-infrared II region fluorescent emission

[0104] The specific method and procedure for preparing a nanohydrogel with neovasculature-targeting near-infrared II region fluorescent emission in this example is the same as in Example 1, except that the 2-carboxyphenylboronic acid used is replaced with an equimolar concentration of 4-carboxyphenylboronic acid.

[0105] The nanohydrogels prepared in Examples 17-22 were detected using the same method as in Examples 1-6.

[0106] The detection results are shown in Table 4.

[0107] Table 4

[0108]

[0109]

[0110] Comparative Example 1, Example 17, Example 18, Example 19 and Example 20, the increase of benzene ring in the precursor molecule did not have a significant impact on the particle size of the nanohydrogel, and due to the conjugation of the benzene ring, the fluorescence emission peak was red-shifted, and the nanohydrogel did not have significant toxicity to HUVEC under light-free conditions, but under light, it could significantly kill HUVEC cells. Comparative Example 1, Example 21 and Example 22, whether the position of the boronic acid group on the benzene ring relative to the carboxyl group, the prepared nanohydrogel can produce near-infrared II region fluorescence, and under light, it can produce a killing effect on HUVEC.

[0111] Example 23, verification of the effect of nanohydrogel-based targeted photodynamic therapy on neovascularization

[0112] A mouse (strain: C57 / BL6j, purchased from Jiangsu Jizhuangkang Biotechnology Co., Ltd.) choroidal neovascularization model was constructed, and 5 μL of the nanohydrogel prepared in Example 1 was injected into the vitreous body of the mouse (a microsyringe was purchased from Hamilton), and after photodynamic therapy, the fluorescence intensity of vascular leakage was detected by fundus fluorescence angiography (Micron IV; Phoenix) using fluorescein sodium as a contrast agent (Abmole).

[0113] Example 24, verification of the effect of nanohydrogel-based targeted photodynamic therapy on neovascularization

[0114] The difference between this example and Example 23 is that the injected nanohydrogel is replaced with a carbon dot dispersion liquid that does not contain fucoidan.

[0115] Table 5

[0116] Test item Example 23 Example 24 Pre-treatment leakage fluorescence intensity values 15000 15580 Post-treatment leakage fluorescence intensity values 100 12000

[0117] Comparative Example 23 and Example 24, when fucoidan is not present, carbon dots do not have neovascular targeting, so the photodynamic process cannot produce a therapeutic effect on neovascularization, and cannot effectively inhibit the leakage caused by neovascularization, and the fluorescence angiography shows strong fluorescence intensity.

[0118] The above detailed description does not constitute a limitation on the scope of protection of the present application. Those skilled in the art should understand that various modifications, combinations, sub-combinations and substitutions can occur depending on design requirements and other factors. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included within the scope of protection of the present application.

Claims

1. A method for the preparation of a nanohydrogel, characterized in that: The method comprises the following steps: (1) dissolving an organic small molecule containing an organic amine and an organic small molecule containing a boric acid group in an acid-containing ethanol solution to obtain a carbon dot precursor solution; the organic small molecule containing an organic amine is selected from at least one of ethylenediamine, aniline, o-phenylenediamine, m-phenylenediamine or p-phenylenediamine; the organic small molecule containing a boric acid group is selected from at least one of 2-carboxyphenylboronic acid, 3-carboxyphenylboronic acid, 4-carboxyphenylboronic acid, phenylboronic acid, 1,4-phenyldiboronic acid or 3-aminophenylboronic acid; (2) obtaining carbon dots by a solvothermal reaction of the carbon dot precursor solution of step (1); (3) adding an ethanol dispersion of the carbon dots of step (2) into a fucoidan solution, and ultrasonicating to obtain a crude product; (4) treating the crude product of step (3) by centrifugation, dialysis and freeze-drying to obtain the nano-hydrogel.

2. The method of claim 1, wherein: In step (1), the concentration of the organic small molecule containing an organic amine in the carbon dot precursor solution is 1 g / L to 10 g / L; and the concentration of the organic small molecule containing a boric acid group is 1 g / L to 10 g / L.

3. The method of claim 1, wherein: In step (1), the molar ratio of the organic small molecule containing an organic amine to the organic small molecule containing a boric acid group in the carbon dot precursor solution is 1:1 to 1:

5.

4. The method of claim 1, wherein: In step (2), the solvothermal reaction is performed in a hydrothermal kettle, the reaction temperature is 140°C to 200°C, and the reaction time is 6 hours to 24 hours.

5. The method of claim 1, wherein: In step (3), the mass percentage concentration of the fucoidan is 0.5% to 3%, and the amount of the carbon dots added is 0.5 g / L to 3 g / L.

6. The method of claim 1, wherein: In step (3), the ethanol dispersion of the carbon dots is added dropwise into the fucoidan solution.

7. The method of claim 1, wherein: In step (4), the centrifugation speed is 5,000 rpm to 8,000 rpm; the dialysis time is 7 days to 14 days, deionized water is used as the dialysis liquid, and the dialysis liquid is replaced once a day; the freeze-drying temperature is -20 to -80°C, and the drying time is 3 to 7 days.

8. The nano-hydrogel prepared by the method of any one of claims 1 to 7.

9. The use of the nano-hydrogel of claim 8 in the preparation of a reagent for fluorescence imaging of ocular fundus neovascularization and a drug for photodynamic therapy of ocular fundus neovascularization.

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