Coating and vascular implant material with coating, preparation method and application thereof

Abstract

The present invention discloses a coating and a vascular implant material with the coating, a preparation method and an application thereof. More specifically, the present invention discloses a coating for coating the surface of a vascular implant material, the coating comprising sulfonated hyaluronic acid / amino-modified carbon dot nanoparticles. Compared with other surface coatings of vascular implant materials that only provide therapeutic functions such as improving biocompatibility, the integrated diagnosis and treatment coating not only gives the material stronger therapeutic functions such as anti-coagulation, anti-inflammation, anti-proliferation and promotion of endothelialization, but also, based on the fluorescent luminescence properties of NP@S-HA / CDs nanoparticles, can accurately locate target cells and organelles, and then diagnose the role of target cells in the pathophysiological conversion of the disease, thereby improving the integrated diagnosis and treatment function of the vascular implant material coating and opening up a broader application prospect for the biochemical surface of vascular implant materials.

Description

Technical Field

[0001] The present invention relates to the field of surface modification of vascular implant materials, and in particular to a coating and a vascular implant material with the coating, as well as a preparation method and application thereof. Background Art

[0002] The surface modification technology of vascular implant materials has gradually matured and is widely used in clinical practice. However, the therapeutic effects of the surfaces of materials currently used in clinical practice are far from the expected goals. The main reason for this is that symptoms such as coagulation, hyperplasia, inflammation and delayed endothelialization on the surface of the materials can lead to problems such as vascular stenosis and restenosis. The basis of new technologies for surface modification of materials is to give the materials good biocompatibility by preparing a multifunctional modification layer on the surface of the materials. The surface coatings of vascular implant materials reported so far mainly improve one or two functions of anticoagulation, antiproliferation, anti-inflammation or endothelialization by inhibiting or promoting the adhesion of cells or proteins on the surface of the materials. The drugs released are mostly traditional inhibitory drugs such as paclitaxel or rapamycin and its derivatives, which do not have good biological activity. Therefore, there is an urgent need to design a vascular implant material coating that can precisely regulate each cell to efficiently and quickly achieve functions such as anticoagulation, antiproliferation, anti-inflammation and promotion of endothelialization. Summary of the Invention

[0003] In order to overcome the deficiencies of the prior art, the present invention provides, in multiple aspects, a coating and a vascular implant material with the coating, as well as a preparation method and application thereof.

[0004] In a first aspect, the present invention provides a coating for coating the surface of a vascular implant material, wherein the coating comprises sulfonated hyaluronic acid / amino-modified carbon dot nanoparticles (NP@S-HA / CDs).

[0005] In a second aspect, the present invention provides a vascular implant material with a coating, comprising a vascular implant material and a coating obtained by the above technical solution, wherein the coating is coated on the surface of the vascular implant material.

[0006] In some embodiments, the vascular implant material includes cobalt-chromium alloy, magnesium alloy, nickel-titanium alloy, or high molecular weight polylactic acid.

[0007] In a third aspect, the present invention provides a method for preparing a coated vascular implant material, comprising the following steps:

[0008] (1) mixing sulfonated hyaluronic acid with an activator to obtain a mixture, adding an amino-modified carbon dot aqueous solution to the mixture to obtain a mixed solution, and drying the mixed solution to obtain sulfonated hyaluronic acid / amino-modified carbon dot nanoparticles; (2) dissolving the sulfonated hyaluronic acid / amino-modified carbon dot nanoparticles in a solvent to obtain a uniform sulfonated hyaluronic acid / carbon dot solution, and coating the sulfonated hyaluronic acid / amino-modified carbon dot solution on the surface of a vascular implant material to obtain a vascular implant material with a coating obtained by the above technical solution.

[0009] In some embodiments, the preparation process of sulfonated hyaluronic acid in step (1) includes: dissolving sodium hyaluronate in deionized water to obtain a mixed solution, adding a strong acid ion exchange resin to the mixed solution, stirring, filtering the strong acid ion exchange resin to obtain a filtrate, adjusting the pH of the filtrate to neutral, and obtaining hyaluronic acid-tetrabutylammonium salt; dissolving the hyaluronic acid-tetrabutylammonium salt and sulfur trioxide pyridine complex in N,N-dimethylformamide solution, stirring under ice bath conditions, then neutralizing the solution, washing to obtain a polymer precipitate, dialyzing the polymer precipitate, and drying to obtain sulfonated hyaluronic acid.

[0010] In some embodiments, the concentration ratio of the added sulfonated hyaluronic acid and amino-modified carbon dot aqueous solution in step (1) is 1-2:1.

[0011] In some embodiments, the preparation process of the activator in step (1) comprises: dissolving 2-(N-morpholino)ethanesulfonic acid monohydrate, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in ultrapure water.

[0012] In some embodiments, the concentration ratio of the 2-(N-morpholino)ethanesulfonic acid monohydrate, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 9.76:0.24:1.

[0013] In some embodiments, the concentration of the sulfonated hyaluronic acid / carbon dot solution in step (2) is 1-10 mg / mL.

[0014] In a fourth aspect, the present invention provides the use of the coated vascular implant material according to the second aspect of the present invention or the coated vascular implant material prepared by the preparation method according to the third aspect of the present invention as a vascular implant.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1. Compared with traditional vascular implant coatings that only regulate cell or protein adsorption on the surface, or release drugs such as paclitaxel or rapamycin and its derivatives, the coating provided by the present invention is an integrated diagnosis and treatment coating that achieves internal cell regulation through the sustained release of nanoparticles, thereby more efficiently imparting anti-coagulation, anti-inflammatory, anti-proliferative and endothelialization functions to the material.

[0017] 2. The preparation process of this integrated diagnosis and treatment coating is simple and easy to operate, does not require expensive and complex equipment, has low process costs, and has significant effects.

[0018] 3. Compared with other vascular implant surface coatings that only provide therapeutic functions such as improving biocompatibility, this integrated diagnosis and treatment coating not only gives the material stronger therapeutic functions such as anti-coagulation, anti-inflammatory, anti-proliferation and promotion of endothelialization, but also, based on the fluorescent luminescence properties of NP@S-HA / CDs nanoparticles, can accurately locate target cells and organelles, and then diagnose the role of target cells in the pathophysiological transformation of the disease, thus improving the integrated diagnosis and treatment function of the vascular implant material coating and opening up broader application prospects for the biochemical surface of vascular implant materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A schematic diagram showing the preparation process of the coated vascular implant material according to Example 1 of the present invention is shown;

[0020] Figure 2 Surface morphology of a vascular implant material without a coating and a vascular implant material with a coating according to an embodiment of the present invention is shown;

[0021] Figure 3 The surface roughness analysis of the vascular implant material without coating and the vascular implant material with coating according to the embodiment of the present invention is shown;

[0022] Figure 4 The present invention shows an embodiment of the present invention for analyzing the functional groups of the surface of the vascular implant material coated with a coating by infrared spectroscopy.

[0023] Figure 5 a shows the fibrinogen adhesion (FGN attachment) test performed on the uncoated vascular implant material and the coated vascular implant material according to the embodiment of the present invention, Figure 5 b shows the denaturation (Confirmational change of FGN) detection of an uncoated vascular implant material and a coated vascular implant material according to an embodiment of the present invention;

[0024] Figure 6a shows the fluorescence staining results of endothelial cells (HUVECs) of the vascular implant material without coating and the vascular implant material with surface coating; Figure 6 b shows the statistical results of the number of endothelial cells (HUVECs) of the vascular implant material without coating and the vascular implant material with surface coating; Figure 6 c shows the nitric oxide release (NO release) test results of the vascular implant material without coating and the vascular implant material with coating on the surface;

[0025] Figure 7 The results of the autofluorescence of nanoparticles in endothelial cells and the staining of lysosomes in endothelial cells of the surface-coated vascular implant material obtained in an embodiment of the present invention are shown;

[0026] Figure 8 The results of smooth muscle cell staining on the surface of the vascular implant material with the surface coating obtained in the embodiment of the present invention are shown;

[0027] Figure 9 a shows the results of macrophage staining on the surface of the vascular implant material coated with a surface coating; Figure 9 b shows the statistical results of the number of macrophages on the surface of the vascular implant material with surface coating. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0029] The present invention provides a coating comprising sulfonated hyaluronic acid / amino-modified carbon dot nanoparticles. Hyaluronic acid is widely present in the extracellular matrix of the human body and other animals and has multiple functions such as regulating cells, delivering factors, and storing water. Studies have found that a coating made of hyaluronic acid with a molecular weight of 1×105DA can simultaneously have multiple functions such as anti-coagulation, anti-inflammation, anti-proliferation, and promoting endothelialization. However, hyaluronidase, which is widely present in the human body, can cut the molecular structure of hyaluronic acid, reduce its molecular weight, and shorten the service life of the hyaluronic acid coating. The sulfonation modification method can not only significantly improve the stability of the hyaluronic acid molecular structure and increase its tolerance to hyaluronidase, but the obtained sulfonated hyaluronic acid (S-HA) also has better anti-coagulation, endothelial promotion, anti-inflammation and anti-proliferation functions than unsulfonated hyaluronic acid (HA). S-HA nano-sizing can improve its delivery efficiency to target cells. Through intracellular regulation after rapid entry into cells, it can more efficiently mobilize cells to achieve established goals, such as anti-coagulation, anti-inflammatory, anti-proliferation, and endothelialization functions on the surface of vascular implant materials. Another advantage of S-HA nano-sizing is that it can be used with electrostatic spraying technology to complete the preparation of vascular implant surface coatings in a few minutes, avoiding the excessive corrosion and structural damage of degradable materials caused by other methods such as self-assembly due to the long preparation time (several hours to dozens of hours). The ethanol solvent used is non-toxic and volatile, and will not cause residue on the material surface.

[0030] The second aspect of the present invention is to provide a vascular implant material with a coating, comprising a vascular implant material and the coating provided by the first aspect of the present invention, wherein the coating is coated on the surface of the vascular implant material. Another drawback of the existing vascular implant material coating is that it is impossible to accurately determine the distribution of the released drug after implantation, such as target cells and organelles. The feedback information is limited, resulting in the inability to accurately optimize and upgrade the designed coating according to the requirements of the lesion site. Amino-modified carbon dots (CDs) and S-HA are nano-processed to synthesize NP@S-HA / CDs nanoparticles with fluorescent luminescence properties, which can accurately determine the whereabouts of the particles and their specific distribution in specific cells after the material is implanted, thereby diagnosing physiological / pathological phenomena at the cellular level.

[0031] The third aspect of the present invention provides a method for preparing a coated vascular implant material, comprising the following steps: (1) mixing sulfonated hyaluronic acid with an activator to obtain a mixture, adding an amino-modified carbon dot aqueous solution to the mixture to obtain a mixed solution, and drying the mixed solution to obtain sulfonated hyaluronic acid / amino-modified carbon dot nanoparticles; (2) dissolving the sulfonated hyaluronic acid / amino-modified carbon dot nanoparticles in a solvent to obtain a uniform sulfonated hyaluronic acid / amino-modified carbon dot solution, and coating the sulfonated hyaluronic acid / amino-modified carbon dot solution on the surface of the vascular implant material to obtain the coated vascular implant material provided by the second aspect of the present invention.

[0032] Preferably, the present invention uses an electrostatic spraying method to graft sulfonated hyaluronic acid and amino-modified carbon dots onto the surface of a vascular implant material to synthesize nanoparticles, thereby producing a coating that efficiently enhances surface anticoagulant, endothelial, anti-inflammatory, and anti-proliferative functions through intracellular regulation of nanoparticles. The NP@S-HA / CDs released from the coating have self-fluorescent properties and can label the regulated cells and their organelles.

[0033] The reaction process and mechanism of the present invention are primarily divided into two parts. The first part involves preparing nanoparticles using sulfonated hyaluronic acid (S-HA) and amino-modified carbon dots (CDs). Using hyaluronic acid (HA) as the starting material, a quaternary ammonium salt is introduced by adding a tetrabutylammonium hydroxide solution to produce the intermediate product, TBA-HA. TBA-HA then reacts with a sulfur trioxide-pyridine complex to yield the final product, S-HA, through a substitution reaction. During the nanoparticle preparation process, S-HA contains carboxyl groups, and CDs contain amino groups, resulting in a chemical reaction of dehydration and condensation to form nanoparticles. The second part involves electrostatic spraying of S-HA / CDs nanoparticles (NP@S-HA / CDs) onto the surface of a vascular implant material. The NP@S-HA / CDs solution to be sprayed is stirred for a period of time to achieve a relatively stable state, then loaded into a syringe. Under the action of a high-voltage electrostatic field, the solution is dispersed into a mist and sprayed out. The solute particles are inertially deposited on the material surface, and the solvent evaporates during the spraying process.

[0034] Surface coating preparation technology is an effective means to improve the biocompatibility of vascular implant materials and enhance their application functions. However, existing coatings and their preparation technologies mainly improve one or two of the following functions: anti-coagulation, anti-proliferation, anti-inflammation, or endothelialization by inhibiting or promoting cell or protein adhesion on the material surface. The released drugs are mostly traditional inhibitory drugs such as paclitaxel or rapamycin and their derivatives, which lack good bioactivity and fall far short of the expected goals. The corrosion-inhibited nanoparticles of the NP@S-HA / CDs coating can not only more efficiently enhance the various functions of vascular implant materials such as anti-coagulation, anti-inflammation, anti-proliferation, and endothelialization through their own intracellular regulation of macrophages, smooth muscle cells, endothelial cells, etc., but can also accurately locate target cells and organelles by virtue of their own fluorescent properties, thereby diagnosing the role of target cells in the pathophysiological transformation of the disease, thus improving the integrated diagnosis and treatment function of the vascular implant material coating.

[0035] In order to make it easier to understand the technical solution of the present application, the following will be described in conjunction with specific examples. The following examples are provided here only for illustrative purposes and should not be construed as specific limitations of the present invention. The experimental methods in the following examples where specific conditions are not mentioned are generally carried out according to conventional experimental conditions or the recommendations of the manufacturer. Reagents or instruments for which the manufacturer or source is not specified are conventional products that can be purchased from the market.

[0036] Example 1

[0037] A vascular implant material with a coating is prepared by the following steps, and the preparation process flow chart is as follows: Figure 1 As shown:

[0038] (1) The molecular weight is 1×10 5 DA sodium hyaluronate is dissolved in deionized water to obtain a hyaluronic acid mixed solution with a concentration of 2 mg / mL. A strong acid ion exchange resin (divinylbenzene) and a sulfonated polymer (styrene and vinylethylbenzene) are added to the mixed solution. The addition ratio of the strong acid ion exchange resin to the hyaluronic acid mixed solution is 10 g of the strong acid ion exchange resin per 100 mL of hyaluronic acid solution. After stirring for 24 hours, the strong acid ion exchange resin is filtered off, and a 10% aqueous solution of tetrabutylammonium hydroxide is added to the filtrate to adjust the pH of the mixed solution to neutral. The mixed solution adjusted to neutrality is then lyophilized to obtain hyaluronic acid-tetrabutylammonium salt, which is then stored in a dark, dry place.

[0039] Hyaluronic acid-tetrabutylammonium salt and sulfur trioxide pyridine complex were dissolved in an N,N-dimethylformamide solution at a molar ratio of 1:8:1. The mixture was stirred in an ice bath for 2 hours. A saturated ethanol solution containing sodium hydroxide was then added to adjust the pH of the solution to neutral. The neutral solution was washed with acetone to obtain a polymer precipitate. The polymer precipitate was dialyzed in deionized water for 5 days, and the dialyzed product was freeze-dried to obtain sulfonated hyaluronic acid (S-HA).

[0040] (2) dissolving 2-(N-morpholino)ethanesulfonic acid monohydrate, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in ultrapure water, wherein the amount of 2-(N-morpholino)ethanesulfonic acid monohydrate is 9.76 mg / mL, N-hydroxysuccinimide is 0.24 mg / mL, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1 mg / mL, to obtain an activator;

[0041] The sulfonated hyaluronic acid obtained in the above step was dissolved in deionized water to obtain a 2 mg / mL sulfonated hyaluronic acid solution. The above activator was mixed with the prepared sulfonated hyaluronic acid solution in a volume ratio of 1:9, and the mixture was evenly shaken in a shaker at 37°C for 30 minutes. Then, a 1 mg / mL amino-modified carbon dot (CDs) aqueous solution was quickly added, wherein (amino-modified carbon dots were purchased from Zhengzhou Feynman Biotechnology Co., Ltd. The volume ratio of the above mixture to the carbon dot aqueous solution was 1:1. After addition, an S-HA / CDs solution was formed (, the S-HA / CDs solution was placed in a vacuum drying oven at 60°C and dried to a viscous state, then placed in a refrigerator at -80°C for 12 hours, and placed in a freeze dryer the next day to be freeze-dried to granules to obtain sulfonated hyaluronic acid / amino-modified carbon dot nanoparticles (NP@S-HA / CDs).

[0042] (3) NP@S-HA / CDs were dissolved in a deionized water solution of ethanol (the volume ratio of ethanol to water was 75%), and vigorously stirred at a certain speed for 2 days at room temperature using a magnetic heating stirrer to obtain a relatively uniform and stable sulfonated hyaluronic acid carbon dot solution (solution concentration 10 mg / mL). The solution was then aspirated into a dedicated syringe, and a high-voltage electrostatic field was formed between the needle and the receiving plate using direct current. When the voltage was about 15 V, the spraying flow rate was 20 μL / min, and the receiving distance was 35 mm, the NP@S-HA / CDs suspension at the needle formed a Taylor stack under the action of the high-voltage electrostatic field, and was atomized and sprayed onto the ZE21B magnesium alloy after the surface anti-corrosion treatment. After 8 minutes of spraying, a coating of NP@S-HA / CDs was obtained on the surface of the ZE21B magnesium alloy, that is, a vascular implant material with a coating, that is, a vascular implant material with a surface coating, which is an integrated diagnosis and treatment coating.

[0043] Example 2

[0044] A vascular implant material with a coating is prepared by the following steps:

[0045] (1) The molecular weight is 1×10 5 DA sodium hyaluronate is dissolved in deionized water to obtain a hyaluronic acid mixed solution with a concentration of 2 mg / mL. A strong acid ion exchange resin (divinylbenzene) and a sulfonated polymer (styrene and vinylethylbenzene) are added to the mixed solution. The addition ratio of the strong acid ion exchange resin to the hyaluronic acid mixed solution is 10 g of the strong acid ion exchange resin per 100 mL of hyaluronic acid solution. After stirring for 24 hours, the strong acid ion exchange resin is filtered off, and a 10% aqueous solution of tetrabutylammonium hydroxide is added to the filtrate to adjust the pH of the mixed solution to neutral. The mixed solution adjusted to neutrality is then lyophilized to obtain hyaluronic acid-tetrabutylammonium salt, which is then stored in a dark, dry place.

[0046] Hyaluronic acid-tetrabutylammonium salt and sulfur trioxide pyridine complex were dissolved in an N,N-dimethylformamide solution at a molar ratio of 1:48:1. The mixture was stirred in an ice bath for 2 hours. A saturated ethanol solution containing sodium hydroxide was then added to adjust the pH of the solution to neutral. The neutral solution was washed with acetone to obtain a polymer precipitate. The polymer precipitate was dialyzed in deionized water for 5 days, and the dialyzed product was freeze-dried to obtain sulfonated hyaluronic acid (S-HA).

[0047] (2) dissolving 2-(N-morpholino)ethanesulfonic acid monohydrate, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in ultrapure water, wherein the amount of 2-(N-morpholino)ethanesulfonic acid monohydrate is 9.76 mg / mL, N-hydroxysuccinimide is 0.24 mg / mL, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1 mg / mL, to obtain an activator;

[0048] The sulfonated hyaluronic acid obtained in the previous step was dissolved in deionized water to obtain a 0.5 mg / mL sulfonated hyaluronic acid solution. The aforementioned activator and the prepared sulfonated hyaluronic acid solution were mixed in a volume ratio of 1:9, and the mixture was shaken evenly in a shaker at 37°C for 30 minutes. Then, a 0.5 mg / mL amino-modified carbon dot (CDs) aqueous solution was quickly added. The amino-modified carbon dots were purchased from Zhengzhou Feynman Biotechnology Co., Ltd. The volume ratio of the above mixture to the carbon dot aqueous solution was 1:1. After addition, an S-HA / CDs solution was formed. The S-HA / CDs solution was placed in a vacuum drying oven at 60°C and dried to a viscous state. It was then placed in a refrigerator at -80°C for 12 hours. The next day, it was placed in a freeze dryer and freeze-dried to granules to obtain sulfonated hyaluronic acid / amino-modified carbon dot nanoparticles (NP@S-HA / CDs).

[0049] (3) NP@S-HA / CDs were dissolved in a deionized water solution of ethanol (the volume ratio of ethanol to water was 50%), and stirred vigorously at a certain speed for 2 days at room temperature using a magnetic heating stirrer to obtain a relatively uniform and stable sulfonated hyaluronic acid carbon dot solution (solution concentration 1 mg / mL). The solution was then aspirated into a dedicated syringe, and a high-voltage electrostatic field was formed between the needle and the receiving plate using DC high voltage electricity. When the voltage was about 15 V, the spraying flow rate was 10 μL / min, and the receiving distance was 35 mm, the NP@S-HA / CDs suspension at the needle formed a Taylor stack under the action of the high-voltage electrostatic field, and was atomized and sprayed onto the cobalt-chromium alloy. After 1 minute of spraying, a coating of NP@S-HA / CDs was obtained on the surface of the cobalt-chromium alloy, that is, a vascular implant material with a coating, that is, a vascular implant material with a surface coating, which is an integrated diagnosis and treatment coating.

[0050] Example 3

[0051] A vascular implant material with a coating is prepared by the following steps:

[0052] (1) The molecular weight is 1×10 5 DA sodium hyaluronate is dissolved in deionized water to obtain a hyaluronic acid mixed solution with a concentration of 2 mg / mL. A strong acid ion exchange resin (divinylbenzene) and a sulfonated polymer (styrene and vinylethylbenzene) are added to the mixed solution. The addition ratio of the strong acid ion exchange resin to the hyaluronic acid mixed solution is 10 g of the strong acid ion exchange resin per 100 mL of hyaluronic acid solution. After stirring for 24 hours, the strong acid ion exchange resin is filtered off, and a 10% aqueous solution of tetrabutylammonium hydroxide is added to the filtrate to adjust the pH of the mixed solution to neutral. The mixed solution adjusted to neutrality is then lyophilized to obtain hyaluronic acid-tetrabutylammonium salt, which is then stored in a dark, dry place.

[0053] Hyaluronic acid-tetrabutylammonium salt and sulfur trioxide pyridine complex were dissolved in N,N-dimethylformamide solution at a molar ratio of 1:36:1. The mixture was stirred in an ice bath for 2 hours. A saturated ethanol solution containing sodium hydroxide was then added to adjust the pH of the solution to neutral. The neutral solution was washed with acetone to obtain a polymer precipitate. The polymer precipitate was dialyzed in deionized water for 5 days, and the dialyzed product was freeze-dried to obtain sulfonated hyaluronic acid (S-HA).

[0054] (2) dissolving 2-(N-morpholino)ethanesulfonic acid monohydrate, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in ultrapure water (wherein 2-(N-morpholino)ethanesulfonic acid monohydrate is 9.76 mg / mL, N-hydroxysuccinimide is 0.24 mg / mL, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1 mg / mL) to obtain an activator;

[0055] The sulfonated hyaluronic acid obtained in the previous step was dissolved in deionized water to obtain a 1 mg / mL sulfonated hyaluronic acid solution. The aforementioned activator was mixed with the prepared sulfonated hyaluronic acid solutions of different concentrations in a volume ratio of 1:9, and the mixture was evenly shaken in a shaker at 37°C for 30 minutes. Then, a 1 mg / mL amino-modified carbon dot (CDs) aqueous solution was quickly added, wherein the amino-modified carbon dots were purchased from Zhengzhou Feynman Biotechnology Co., Ltd. The volume ratio of the aforementioned mixture to the carbon dot aqueous solution was 1:1. After addition, an S-HA / CDs solution was formed. The S-HA / CDs solution was placed in a vacuum drying oven at 60°C and dried to a viscous state. It was then placed in a refrigerator at -80°C for 12 hours and freeze-dried in a freezer the next day until granular, obtaining sulfonated hyaluronic acid / amino-modified carbon dot nanoparticles (NP@S-HA / CDs).

[0056] (3) NP@S-HA / CDs were dissolved in a deionized water solution of ethanol (the volume ratio of ethanol to water was 50%), and vigorously stirred at a certain speed for 3 days at room temperature using a magnetic heating stirrer to obtain a relatively uniform and stable sulfonated hyaluronic acid carbon dot solution (solution concentration 5 mg / mL), which was then aspirated into a dedicated syringe. A high-voltage electrostatic field was formed between the needle and the receiving plate using direct current. When the voltage was about 15 V, the spraying flow rate was 15 μL / min, and the receiving distance was 35 mm, the NP@S-HA / CDs suspension at the needle formed a Taylor stack under the action of the high-voltage electrostatic field, and was atomized and sprayed onto the polymer polylactic acid. After 4 minutes of spraying, a coating of NP@S-HA / CDs was obtained on the surface of the polymer polylactic acid, that is, a vascular implant material with a coating, that is, a vascular implant material with a surface coating, which is an integrated diagnosis and treatment coating.

[0057] Example 4

[0058] A vascular implant material with a coating is prepared by the following steps:

[0059] (1) The molecular weight is 1×10 5DA sodium hyaluronate is dissolved in deionized water to obtain a hyaluronic acid mixed solution with a concentration of 2 mg / mL. A strong acid ion exchange resin (divinylbenzene) and a sulfonated polymer (styrene and vinylethylbenzene) are added to the mixed solution. The addition ratio of the strong acid ion exchange resin to the hyaluronic acid mixed solution is 10 g of the strong acid ion exchange resin per 100 mL of hyaluronic acid solution. After stirring for 24 hours, the strong acid ion exchange resin is filtered off, and a 10% aqueous solution of tetrabutylammonium hydroxide is added to the filtrate to adjust the pH of the mixed solution to neutral. The mixed solution adjusted to neutrality is then lyophilized to obtain hyaluronic acid-tetrabutylammonium salt, which is then stored in a dark, dry place.

[0060] Hyaluronic acid-tetrabutylammonium salt and sulfur trioxide pyridine complex were dissolved in N,N-dimethylformamide solution at a molar ratio of 1:27:1. The mixture was stirred in an ice bath for 2 hours. A saturated ethanol solution containing sodium hydroxide was then added to adjust the pH of the solution to neutral. The neutral solution was washed with acetone to obtain a polymer precipitate. The polymer precipitate was dialyzed in deionized water for 5 days, and the dialyzed product was freeze-dried to obtain sulfonated hyaluronic acid (S-HA).

[0061] (2) dissolving 2-(N-morpholino)ethanesulfonic acid monohydrate, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in ultrapure water, wherein the amount of 2-(N-morpholino)ethanesulfonic acid monohydrate is 9.76 mg / mL, N-hydroxysuccinimide is 0.24 mg / mL, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1 mg / mL, to obtain an activator;

[0062] The sulfonated hyaluronic acid obtained in the previous step was dissolved in deionized water to obtain a 1.5 mg / mL sulfonated hyaluronic acid solution. The aforementioned activator and the prepared sulfonated hyaluronic acid solution were mixed in a volume ratio of 1:9, and the mixture was shaken evenly in a shaker at 37°C for 30 minutes. Then, a 1 mg / mL aqueous solution of amino-modified carbon dots (CDs) was quickly added. The amino-modified carbon dots were purchased from Zhengzhou Feynman Biotechnology Co., Ltd. The volume ratio of the aforementioned mixture to the carbon dot aqueous solution was 1:1. After addition, an S-HA / CDs solution was formed. The S-HA / CDs solution was placed in a vacuum drying oven at 60°C to dry until viscous, then placed in a -80°C refrigerator for 12 hours. The next day, it was placed in a freeze dryer and freeze-dried to granules to obtain sulfonated hyaluronic acid / amino-modified carbon dot nanoparticles (NP@S-HA / CDs).

[0063] (3) NP@S-HA / CDs were dissolved in a deionized water solution of ethanol (the volume ratio of ethanol to water was 100%), and vigorously stirred at a certain speed for 2 days at room temperature using a magnetic heating stirrer to obtain a relatively uniform and stable sulfonated hyaluronic acid carbon dot solution (solution concentration 8 mg / mL), which was then aspirated into a dedicated syringe. A high-voltage electrostatic field was formed between the needle and the receiving plate using direct current. When the voltage was about 15 V, the spraying flow rate was 20 μL / min, and the receiving distance was 35 mm, the NP@S-HA / CDs suspension at the needle formed a Taylor stack under the action of the high-voltage electrostatic field, and was atomized and sprayed onto the nickel-titanium alloy after the surface anti-corrosion treatment. After 6 minutes of spraying, a coating of NP@S-HA / CDs was obtained on the surface of the nickel-titanium alloy, that is, a vascular implant material with a coating, that is, a vascular implant material with a surface coating, which is an integrated diagnosis and treatment coating.

[0064] The physicochemical properties of the coated vascular implant materials obtained in Examples 1 to 4 were further analyzed. The results showed that the physicochemical properties of the coated vascular implant materials obtained in Examples 1 to 4 were similar. Only the relevant data of Example 1 are shown in the present invention. Figure 2 The surface morphology of the vascular implant material without coating and the vascular implant material with coating is shown in FIG. Figure 2 As shown, evenly distributed particles can be seen on the surface of the vascular implant material coated with the coating, indicating that the preparation method of the present invention successfully coats the surface of the vascular implant material with a coating.

[0065] Furthermore, the present invention analyzes the surface roughness of the vascular implant material coated with the coating. Figure 3 The surface roughness analysis of vascular implant materials without coating and vascular implant materials with coating is shown. Figure 3 As shown, the surface of the vascular implant material coated with the coating is rougher than the surface of the vascular implant material not coated with the coating.

[0066] Furthermore, the present invention analyzes the infrared spectrum detection functional groups on the surface of the vascular implant material coated with the coating. Figure 4 As shown, compared with the uncoated vascular implant material, the infrared spectrum of the coated vascular implant material has a characteristic peak at a wavelength of about 900 nm, indicating that the coated vascular implant material has sulfonic acid groups (SO2) present, proving that the present invention has successfully coated the sulfonated hyaluronic acid / amino-modified carbon dot nanoparticles on the surface of the vascular implant material.

[0067] Fibrinogen adhesion and denaturation are the precursor events of coagulation and thrombosis. By inhibiting the adhesion and denaturation of fibrinogen, thrombosis can be effectively prevented, thereby preventing blood vessel blockage. Figure 5a shows the fibrinogen adhesion (FGN attachment) test performed on the uncoated vascular implant material and the coated vascular implant material according to the embodiment of the present invention, Figure 5 b shows the denaturation (Confirmational change of FGN) test of the vascular implant material without coating and the vascular implant material with coating according to the embodiment of the present invention. Figure 5 a and Figure 5 As shown in Figure b, compared with the vascular implant material not coated with the coating, the amount of fibrinogen adhesion and denaturation on the surface of the vascular implant material coated with the coating is significantly reduced, indicating that coating the surface of the vascular implant material with the coating of the present invention can significantly inhibit fibrinogen adhesion and denaturation.

[0068] Endothelial cells are important components of the vascular endothelium and play an irreplaceable role in vascular tissue repair, functional maintenance, and endothelialization of vascular implant surfaces. The present invention further verifies the adhesion and proliferation of endothelial cells. Figure 6 a. Figure 6 b. Figure 6 Figure c shows the fluorescence staining, number counting, and nitric oxide (NO) release assay results of endothelial cells (HUVECs) for the uncoated vascular implant material and the surface-coated vascular implant material, respectively. The results show that the surface-coated vascular implant material can significantly promote the adhesion, proliferation, and NO release of surface endothelial cells.

[0069] Figure 7 The results of the autofluorescence of nanoparticles and the staining of lysosomes in the endothelial cells of the vascular implant material of the surface coating obtained in the embodiment of the present invention are shown, wherein the autofluorescence of nanoparticles is blue light and the fluorescence of lysosomes is purple light. Lysosomes are organelles in cells that are responsible for digesting and metabolizing foreign particles engulfed by them. The results show that although the nanoparticles partially overlap with the lysosomes, they are not concentrated in the lysosomes, but are evenly distributed throughout the cell. They will not be digested and metabolized by the lysosomes in the short term. The particles enter the vascular endothelial cells very quickly. Within 2-6 hours, a large number of particles can be seen evenly distributed in the cells, which is conducive to the coating's more efficient improvement of surface anti-coagulation, anti-inflammatory, anti-proliferation and endothelialization functions.

[0070] Figure 8The results of smooth muscle cell staining on the surface of the vascular implant material with the surface coating obtained in the embodiment of the present invention are shown. Excessive proliferation of smooth muscle cells can easily lead to hyperplasia on the surface of the vascular material, thereby causing restenosis in the blood vessel. Therefore, inhibiting excessive proliferation of smooth muscle cells on the surface of the material is one of the main goals of designing a coating on the surface of the vascular implant material. The results show that the number of smooth muscle cells on the surface of the vascular implant material with the coating obtained in the embodiment of the present invention is significantly less than the number of smooth muscle cells on the surface of the vascular implant material without the coating, and the coating of the embodiment of the present application can significantly improve the anti-proliferative function of the surface of the vascular implant material.

[0071] Figure 9 a. Figure 9 Figure b shows the results of macrophage staining and cell count on the surface of a coated vascular implant. Macrophages are key players in inflammation, and inflammatory responses can delay endothelialization on the surface of vascular implants, increase the risk of proliferation, and shorten the material's service life. The results show that the number of macrophages on the coated surface of the vascular implant is significantly lower than that on the uncoated surface, demonstrating that the coatings of the present invention significantly enhance the anti-inflammatory properties of the vascular implant surface.

[0072] The above embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and replacements made by technicians in this field on the basis of the present invention fall within the scope of protection required by the present invention.

Claims

1. A method for preparing a coated vascular implant material, characterized in that: The following steps are involved: (1) mixing sulfonated hyaluronic acid and an activator to obtain a mixture, adding an amino-modified carbon dot aqueous solution to the mixture to obtain a mixed solution, and drying the mixed solution to obtain sulfonated hyaluronic acid / amino-modified carbon dot nanoparticles; (2) dissolving the sulfonated hyaluronic acid / amino-modified carbon dot nanoparticles in a solvent to obtain a uniform sulfonated hyaluronic acid / amino-modified carbon dot solution, and coating the sulfonated hyaluronic acid / carbon dot solution on the surface of a vascular implant material to obtain a vascular implant material coated with the sulfonated hyaluronic acid / carbon dot solution.

2. The method according to claim 1, wherein The preparation process of sulfonated hyaluronic acid in step (1) comprises: dissolving sodium hyaluronate in deionized water to obtain a mixed solution, adding a strong acid ion exchange resin to the mixed solution, stirring, filtering the strong acid ion exchange resin to obtain a filtrate, and adjusting the pH of the filtrate to neutral to obtain hyaluronic acid-tetrabutylammonium salt; The hyaluronic acid-tetrabutylammonium salt and sulfur trioxide pyridine complex is dissolved in N,N-dimethylformamide solution and stirred under ice bath conditions. The solution is then neutralized and washed to obtain a polymer precipitate. The polymer precipitate is dialyzed and dried to obtain sulfonated hyaluronic acid.

3. The method according to claim 1, wherein In the step (1), the added concentration ratio of sulfonated hyaluronic acid to amino-modified carbon dot aqueous solution is 1-2:

1.

4. The method according to claim 1, wherein The preparation process of the activator in step (1) comprises: dissolving 2-(N-morpholino)ethanesulfonic acid monohydrate, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in ultrapure water.

5. The method according to claim 4, wherein The concentration ratio of the 2-(N-morpholino)ethanesulfonic acid monohydrate, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 9.76:0.24:

1.

6. The method according to claim 1, wherein The concentration of the sulfonated hyaluronic acid / carbon dot solution in step (2) is 1-10 mg / mL.

7. A coating for coating the surface of a vascular implant material, characterized in that: Prepared by the method according to any one of claims 1 to 6, the coating comprises sulfonated hyaluronic acid / amino-modified carbon dot nanoparticles.

8. A coated vascular implant material, characterized in that: The invention comprises a vascular implant material and the coating according to claim 7, wherein the coating is coated on the surface of the vascular implant material.

9. The vascular implant material according to claim 8, wherein: The vascular implant material includes cobalt-chromium alloy, magnesium alloy, nickel-titanium alloy or high molecular weight polylactic acid.

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