Exclusive Coating for Fully Degradable Magnesium Alloy Vascular Stent Material, Preparation Method Thereof, and Application

Abstract

The present invention belongs to the field of surface coatings and modification materials for magnesium alloy vascular stents, and particularly relates to an exclusive coating for a fully degradable magnesium alloy vascular stent material, a preparation method thereof, and an application. The preparation method of the exclusive coating for the fully degradable magnesium alloy vascular stent material of the present invention first prepares a Schiff base mixed solution and a nanoparticle solution of sulfonated hyaluronic acid, and then deposits the Schiff base on the magnesium alloy surface by electrostatic spraying to form a dense coating with a degradation inhibition function, and then sprays and grafts sulfonated hyaluronic acid nanoparticles on the surface of the Schiff base coating to form a Schiff base-sulfonated hyaluronic acid nanoparticle composite coating. The exclusive coating for the magnesium alloy vascular stent material obtained by the above method of the present invention can inhibit the degradation of the magnesium alloy matrix, endow the magnesium alloy stent material with comprehensive functions of anticoagulation, anti-inflammation, endothelialization, anti-proliferation, and intracellular delivery of magnesium ions, and has good application prospects in the field of coating materials for magnesium alloy stents.

Description

Technical Field

[0001] The present invention belongs to the field of surface coatings and modification materials for magnesium alloy vascular stents, and particularly relates to an exclusive coating for a fully degradable magnesium alloy vascular stent material, a preparation method thereof, and an application thereof. Background Art

[0002] As a vascular stent material, degradable magnesium alloy has good degradability, biocompatibility, safety, and excellent mechanical properties. In recent years, it has been gradually tested and promoted clinically and has become an ideal material for replacing traditional stents in the treatment of cardiovascular diseases.

[0003] With the in-depth research and application of magnesium alloy in vascular stents, as a material with high bioactivity, the problems of too fast degradation and delayed endothelialization of magnesium alloy have become technical bottlenecks restricting its further application. Complications such as thrombosis, inflammation, and hyperplasia caused thereby also seriously affect the physical health and quality of life of patients.

[0004] A large number of studies have shown that surface coating preparation is an effective means to effectively inhibit the degradation of magnesium alloy used in vascular stents, or endow it with functions such as endothelialization, anti-thrombosis, anti-inflammation, and anti-hyperplasia. However, the coatings of magnesium alloy stents developed at the present stage all have problems such as excessive corrosion of the substrate due to too long preparation time, cytotoxicity of the degradation inhibitor in the coating, and too thick generated intima or delayed endothelialization.

[0005] At present, the coating of the DREAMS series of magnesium alloy stents of Biotronik company used in clinical trials is made of poly(lactic acid) loaded with rapamycin and its derivatives, which can effectively inhibit hyperplasia, but still does not solve the problems of too fast degradation and delayed endothelialization. And various coatings being developed by different biological companies at the present stage, such as fluorination, alkali heat passivation, and the assembly of other bioactive substances such as proteins and polypeptides, are time-consuming and easily cause excessive corrosion of magnesium alloy stents, shortening their service life and reducing their service functions.

[0006] In view of the problems in the prior art, it is urgent to develop a special coating suitable for degradable magnesium alloy, which can be prepared on the magnesium substrate surface in an extremely short time to avoid excessive corrosion, has high safety, the degradation inhibitor has no cytotoxicity, and can also adapt the degradation rate of magnesium alloy to behaviors such as promoting endothelialization, so as to better meet the application requirements of degradable magnesium alloy stents. Summary of the Invention

[0007] The first object of the present invention is to provide a preparation method for a special coating of a fully degradable magnesium alloy vascular stent material. By this method, a coating can be formed on the magnesium alloy surface in an extremely short time, avoiding excessive corrosion of the magnesium alloy during the coating preparation process; and through the biological modification of the magnesium alloy, the degradation of the magnesium alloy matrix can be effectively inhibited, while loading the degraded magnesium ions into cells for intracellular regulation, which can endow the fully degradable magnesium alloy vascular stent with anticoagulant, anti-inflammatory, anti-proliferative, and endothelialization-promoting functions.

[0008] The second object of the present invention is to provide a special coating for a fully degradable magnesium alloy vascular stent material, which can inhibit the degradation of the magnesium alloy matrix, can also promote the rapid endothelialization of the magnesium alloy material surface, and has anticoagulant, anti-inflammatory, and anti-proliferative functions, and is suitable for use in magnesium alloy stents.

[0009] The third object of the present invention is to provide an application of a special coating for a fully degradable magnesium alloy vascular stent material.

[0010] In order to achieve the above objects, the technical solution adopted for the preparation method of the special coating of the fully degradable magnesium alloy vascular stent material of the present invention is as follows:

[0011] A preparation method for a special coating of a fully degradable magnesium alloy vascular stent material includes the following steps:

[0012] (1) Dissolve methionine Schiff base, glycine Schiff base, and lysine Schiff base in an organic solvent to obtain a Schiff base mixed solution;

[0013] React sodium hyaluronate with a quaternary ammonium salt to obtain a quaternary ammonium salt of hyaluronic acid; then carry out a substitution reaction between the quaternary ammonium salt of hyaluronic acid and a sulfur trioxide pyridine complex to obtain sulfonated hyaluronic acid; in the presence of an activator, react sulfonated hyaluronic acid with polyethyleneimine to obtain a nanoparticle solution of sulfonated hyaluronic acid;

[0014] (2) Using the electrostatic spraying method, first spray the Schiff base mixed solution on the surface of the magnesium alloy substrate, and then spray the nanoparticle solution of sulfonated hyaluronic acid after curing to obtain a special coating for the fully degradable magnesium alloy vascular stent material.

[0015] The reaction process and mechanism of the preparation method for the special coating of the fully degradable magnesium alloy vascular stent material provided by the present invention are mainly divided into two steps:

[0016] The first step is the preparation of the Schiff base mixture and sulfonated hyaluronic acid (S-HA) nanoparticle solution. The Schiff base mixture is prepared by dissolving three different Schiff bases in a solvent to obtain a mixture containing the three Schiff bases. The preparation of the S-HA nanoparticle solution uses hyaluronic acid (HA) as the raw material. By adding a quaternary ammonium salt, an intermediate product, tetrabutylammonium hyaluronate (TBA-HA), is obtained. Then, TBA-HA reacts with pyridine sulfur trioxide complex, and through a substitution reaction, the product sulfonated hyaluronic acid (S-HA) is obtained. By adjusting the ratio of TBA-HA to pyridine sulfur trioxide complex in this process, S-HA with different sulfur contents can be obtained. During the preparation of the nanoparticles, S-HA is negatively charged due to the presence of carboxyl groups, and polyethyleneimine (PEI) is positively charged due to the presence of amino groups. Therefore, the two can interact through chemical reactions to form nanoparticles.

[0017] The second step is the ordered electrostatic spraying of Schiff base and S-HA nanoparticles on the surface of the magnesium alloy substrate. First, the Schiff base mixture is sprayed on the surface of the magnesium alloy substrate. After curing, the S-HA nanoparticle solution is sprayed. Under the action of a high-voltage electrostatic field, the solution is broken into a mist and sprayed out. The solute particles are deposited on the material surface by inertia, and the solvent volatilizes during the spraying process, thus obtaining a composite coating of Schiff base-sulfonated hyaluronic acid nanoparticles.

[0018] In the above preparation method of the present invention, a composite coating formed by Schiff base and sulfonated hyaluronic acid nanoparticles is used to perform biocompatible modification on the magnesium alloy. Among them, Schiff base is a class of organic compounds containing imine (-C=N) or methylimine characteristic groups. Due to the C=N double bond it contains, it can bind with Mg 2+ ions to form a stable complex. Therefore, the present invention uses Schiff base to complex with magnesium ions generated by the degradation of the magnesium alloy to form a dense complex passivation layer to inhibit the degradation of the magnesium alloy. Sulfonated hyaluronic acid (S-HA) nanoparticles can not only regulate macrophages, smooth muscle cells, endothelial cells, etc. intracellularly by themselves to improve various functions of the magnesium alloy such as anticoagulation, anti-inflammation, anti-proliferation, and endothelialization, but also bind with some magnesium ions to transport magnesium elements into macrophages and endothelial cells, further enhancing the anti-inflammatory and endothelialization functions of the stent. Moreover, in the present invention, the Schiff base and S-HA nanoparticles are sequentially sprayed on the surface of the magnesium alloy substrate material by electrostatic spraying method in a very short time, which will not cause excessive corrosion of the stent, and at the same time can take into account the degradation inhibitor of the stent and various functions such as anticoagulation, anti-inflammation, anti-proliferation, and endothelialization. Therefore, the preparation method of the present invention discloses a brand-new preparation idea for magnesium alloy coatings and is a new method suitable for preparing exclusive functional active coatings on the surface of fully degradable magnesium alloy vascular stent materials.

[0019] In step (1), preferably, the methionine Schiff base is prepared by subjecting methionine and paeonol to a thermal reflux reaction; the glycine Schiff base is prepared by subjecting glycine and paeonol to a thermal reflux reaction; the lysine Schiff base is prepared by subjecting lysine and paeonol to a thermal reflux reaction. The Schiff base synthesized from the natural product amino acid and paeonol in the present invention has no cytotoxicity and has good biocompatibility. Therefore, it can be used as a degradation inhibitor for the exclusive coating of magnesium alloys for vascular stents.

[0020] Furthermore, the temperature of the thermal reflux reaction is 60 - 80°C, and the time of the thermal reflux reaction is 1.5 - 3 h.

[0021] Preferably, in step (1), the organic solvent is absolute ethanol. The ethanol solvent used in the present invention is non-toxic and volatile, and will not remain on the surface of the stent.

[0022] In the Schiff base mixture, the molar concentrations of the methionine Schiff base, glycine Schiff base, and lysine Schiff base are 0.04 M, 0.02 M, and 0.02 M in sequence.

[0023] Hyaluronic acid widely exists in the extracellular matrix of human and other animal cells and has various functions such as regulating cells, delivering factors, and storing water. The functions of hyaluronic acid with different molecular weights are completely different. Research and development have found that although all molecular weights of hyaluronic acid have the function of inhibiting platelet adhesion and thus obtaining anticoagulant function, low molecular weight hyaluronic acid (4×10 3 Da and below) is prone to induce inflammation, and the coating made of high molecular weight hyaluronic acid (5×10 5 Da and above) is not conducive to the adhesion and proliferation of endothelial cells. Only the coating made of medium molecular weight hyaluronic acid (around 1×10 5 Da) can simultaneously have multiple functions such as anticoagulation, anti-inflammation, anti-proliferation, and promoting endothelialization. Therefore, preferably, in step (1), the molecular weight of the sodium hyaluronate is 1×10 5 ~2×10 5 Da.

[0024] Furthermore, the quaternary ammonium salt is tetrabutylammonium hydroxide.

[0025] When the hyaluronic acid quaternary ammonium salt and the sulfur trioxide pyridine complex carry out a substitution reaction, the mass ratio of the hyaluronic acid quaternary ammonium salt to the sulfur trioxide pyridine complex is 1:(8 - 48).

[0026] The role of the activator is to activate the carboxyl groups in sulfonated hyaluronic acid so that the carboxyl groups of sulfonated hyaluronic acid and the amino groups of polyethyleneimine can effectively react. Preferably, the activator is a ternary combination of sodium methyl ester sulfonate (MES), N-hydroxysuccinimide (NHS), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC).

[0027] The process of preparing sulfonated hyaluronic acid from sodium hyaluronate can adopt conventional methods in the art, and the present invention does not make special limitations. Specifically, the following steps can be adopted: Dissolve sodium hyaluronate in ultrapure water, then add strong acid cation exchange resin, stir, stand, filter, then add quaternary ammonium salt solution until alkaline, freeze-dry, dissolve the obtained products, hyaluronic acid quaternary ammonium salt and sulfur trioxide pyridine complex, respectively, in N,N-dimethylformamide solution, then mix and stir the two under ice bath conditions, add saturated sodium hydroxide ethanol solution, adjust the pH of the mixed system to 8.0, wash with acetone, dialyze the obtained precipitate in ultrapure water, and freeze-dry to obtain sulfonated hyaluronic acid. At present, hyaluronidase widely present in the human body can shear the molecular structure of hyaluronic acid, reduce its molecular weight, and shorten the service life of the hyaluronic acid coating. However, through sulfonation modification, the present invention can not only significantly improve the stability of the hyaluronic acid molecular structure and its tolerance to hyaluronidase, but also particularly find that the obtained sulfonated hyaluronic acid (S-HA) has better anticoagulant, pro-endothelial, anti-inflammatory, and anti-proliferative functions compared with un-sulfonated hyaluronic acid (HA).

[0028] Further, in step (1), the reaction between sulfonated hyaluronic acid and polyethyleneimine is as follows: Prepare an aqueous solution of sulfonated hyaluronic acid at a concentration of 2 mg / mL, then add an aqueous solution of polyethyleneimine at 0.5 mg / mL after shaking for a period of time under the condition of 25-40 °C to obtain a nanoparticle solution of sulfonated hyaluronic acid. The present invention further nano-modifies S-HA to improve its delivery efficiency to target cells. Through intracellular regulation after quickly entering cells, it can more efficiently mobilize cells to achieve the established goals and improve the functions of magnesium alloy for vascular stents such as anticoagulation, anti-inflammation, anti-proliferation, and endothelialization. In addition, S-HA nanoparticles can also bind to magnesium ions in the degradation products of magnesium alloy through charge effect, deliver magnesium ions into macrophages and endothelial cells, and further improve the anti-inflammatory and endothelialization functions of the coating.

[0029] Preferably, the volume ratio of the aqueous solution of sulfonated hyaluronic acid to the aqueous solution of polyethyleneimine is (9-11):1.

[0030] During spraying in step (2), the concentration of the nanoparticle solution of sulfonated hyaluronic acid is 8 mg / mL.

[0031] In step (2), the voltage for spraying is 9 - 11 kV, the spraying flow rate is 15 - 21 μm / min, and the spraying time is 1.0 - 1.5 min. Another prominent advantage of the S-HA nanocrystallization of the present invention is that, by means of electrostatic spraying technology, the preparation of the surface coating of the magnesium alloy stent can be completed within a few minutes, avoiding the excessive corrosion and structural damage of the magnesium alloy stent caused by the overly long preparation time (from several hours to dozens of hours) of other methods such as self-assembly in the past.

[0032] The exclusive coating of the fully degradable magnesium alloy vascular stent material of the present invention is prepared by the preparation method as described above.

[0033] In the present invention, a Schiff base coating and a sulfonated hyaluronic acid nanoparticle coating are successively grafted onto the surface of a magnesium alloy substrate by electrostatic spraying. The Schiff base coating and the sulfonated hyaluronic acid nanoparticle coating are tightly combined through electrostatic adsorption, hydrogen bonding, van der Waals forces, and physical adsorption. Moreover, the coating components will slowly degrade and release together with the magnesium alloy. The obtained modified layer can take into account the multiple functions of inhibiting the degradation of magnesium alloy and improving surface anticoagulation, promoting endothelium, anti-inflammation, and anti-proliferation, and is very suitable for use as the exclusive functional active coating of magnesium alloy vascular stents.

[0034] The present invention also provides the application of the exclusive coating of the fully degradable magnesium alloy vascular stent material as described above, specifically the application in the preparation of a functional coating for a fully degradable magnesium alloy vascular stent.

[0035] Furthermore, the present invention does not make special limitations on the material of the magnesium alloy vascular stent, and any magnesium alloy material conventionally used in the art and suitable for use as a magnesium alloy vascular stent can be adopted. For example, the magnesium alloy can be a ZE21B magnesium alloy, a Mg-Zn-Y-Nd magnesium-based alloy, etc.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] (1) The preparation of the surface coating is the key technology to solve the problems of too fast degradation and delayed endothelialization of fully degradable magnesium alloy vascular stents. For the exclusive coating of the fully degradable magnesium alloy vascular stent material of the present invention, the Schiff base can complex with the magnesium ions generated by the degradation of the magnesium alloy to form a dense complex passivation layer; the S-HA nanoparticles can not only regulate macrophages, smooth muscle cells, endothelial cells, etc. intracellularly by themselves to improve various functions such as anticoagulation, anti-inflammation, anti-proliferation, and endothelialization of the magnesium alloy, but also combine with some magnesium ions to transport magnesium elements into macrophages and endothelial cells, further strengthening the anti-inflammatory and endothelialization functions of the stent.

[0038] (2) The preparation process of the exclusive coating of the fully degradable magnesium alloy vascular stent material is simple and easy to operate, does not require expensive and complex equipment, has a low process cost, and is suitable for industrial application.

[0039] (3) Compared with the preparation time of several hours to dozens of hours for other existing coating preparation technologies, the Schiff base / sulfonated hyaluronic acid nanoparticle composite coating of the present invention is prepared by electrostatic spraying method in an extremely short time. The coating preparation process will not cause excessive corrosion of the magnesium alloy, and after spraying, it will not cause excessive corrosion of the magnesium alloy stent, shortening its service life and reducing its service function. Moreover, the obtained composite coating can significantly inhibit the degradation, anti-thrombosis, anti-inflammation, anti-proliferation and endothelialization promotion functions of the magnesium alloy.

[0040] Therefore, the exclusive coating for the fully degradable magnesium alloy vascular stent material of the present invention has broad application prospects in the field of surface coatings for fully degradable magnesium alloy vascular stent materials. Brief Description of the Drawings

[0041] Figure 1 is the process flow chart of the preparation method of the exclusive coating for the fully degradable magnesium alloy vascular stent material of the present invention;

[0042] Figure 2 is the scanning electron microscope image of the magnesium alloy substrate sample for vascular stent without coating (left figure) in Example 1 of the present invention and the Schiff base-S-HA nanoparticle coated magnesium alloy sample prepared in Example 1 (right figure);

[0043] Figure 3 is the corrosion morphology image of the magnesium alloy substrate sample for vascular stent without coating (left figure) in Example 1 of the present invention and the Schiff base-S-HA nanoparticle coated magnesium alloy sample prepared in Example 1 (right figure) after being soaked in human blood for 7 days;

[0044] Figure 4 is the fluorescence staining result image of endothelial cells on the surface of the magnesium alloy substrate sample for vascular stent without coating (left figure) in Example 1 of the present invention and the Schiff base-S-HA nanoparticle coated magnesium alloy sample prepared in Example 1 (right figure);

[0045] Figure 5 is the light microscope image (Figure a) of vascular endothelial cells migrating on the surface of the magnesium alloy substrate sample for vascular stent without coating in Example 1 of the present invention and the Schiff base-S-HA nanoparticle coated magnesium alloy sample prepared in Example 1 and the statistical result image of the migration distance (Figure b);

[0046] Figure 6 is the culture result image of smooth muscle cells on the surface of the magnesium alloy substrate sample for vascular stent without coating in Example 1 of the present invention and the Schiff base-S-HA nanoparticle coated magnesium alloy sample prepared in Example 1; among them, Figure a is the fluorescence staining image of smooth muscle cells; Figure b is the three-dimensional imaging diagram of the fluorescence intensity Ipwin32 of α-SMA;

[0047] Figure 7Results of macrophage culture on the magnesium alloy substrate sample without coating for the vascular stent in Example 1 of the present invention and the Schiff base-S-HA nanoparticle-coated magnesium alloy sample prepared in Example 1; wherein Figure a is the fluorescence staining map of macrophages, and Figure b is the three-dimensional imaging map of the fluorescence intensities of TNF-α and CD206 Ipwin32.

[0048] Figure 8 Morphology and number of platelets adhered to the surface of the magnesium alloy substrate sample (left figure) without coating for the vascular stent in Example 1 of the present invention and the Schiff base-S-HA nanoparticle-coated magnesium alloy sample (right figure) prepared in Example 1 after being immersed in human platelet-rich plasma for 1 hour. Specific Embodiments

[0049] The technical solution of the present invention will be further described below in conjunction with specific embodiments, but it does not constitute a limitation to the present invention. The specific conditions not described in the following examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or raw materials used in the following examples are all conventional products obtained through commercial channels.

[0050] In the following examples, the preparation method of the exclusive coating for the fully degradable magnesium alloy vascular stent material provided by the present invention has a process flow diagram as Figure 1 shown. The process flow will be introduced below in conjunction with specific examples.

[0051] Example 1

[0052] This example provides a preparation method for the exclusive coating of the fully degradable magnesium alloy vascular stent material. The magnesium alloy substrate used is ZE21B magnesium alloy.

[0053] To prepare a Schiff base / sulfonated hyaluronic acid nanoparticle coating on the surface of ZE21B magnesium alloy, the specific steps are as follows:

[0054] (1) Dissolve 0.01 mol of methionine in 60 mL of absolute ethanol to obtain an ethanol solution of methionine, dissolve 0.01 mol of paeonol in 15 mL of absolute ethanol to obtain a paeonol solution, and gradually add the ethanol solution of methionine dropwise to the paeonol solution. Reflux at 60 °C for 3 h, then cool, filter, and dry to obtain methionine Schiff base; then use glycine and paeonol, lysine and paeonol respectively, and refer to the preparation process and parameters of methionine Schiff base to prepare glycine Schiff base and lysine Schiff base.

[0055] Weigh methionine Schiff base, glycine Schiff base, and lysine Schiff base respectively and dissolve them in absolute ethanol, then stir to make a compound Schiff base mixture solution for standby; in the Schiff base mixture solution, the molar concentrations of methionine Schiff base, glycine Schiff base, and lysine Schiff base are 0.04 M, 0.02 M, and 0.02 M in sequence.

[0056] (2) After fully dissolving sodium hyaluronate with a molecular weight of 1×10 5 Da in ultrapure water (2 mg / mL), add strongly acidic ion exchange resin (the dosage of strongly acidic ion exchange resin corresponding to every 100 mL of sodium hyaluronate solution is 10 g), stir, let stand, filter, then add tetrabutylammonium hydroxide aqueous solution (concentration 10%) to make it alkaline, and freeze-dry to obtain quaternary ammonium salt of hyaluronic acid; dissolve the quaternary ammonium salt of hyaluronic acid and sulfur trioxide pyridine complex with a mass ratio of 1:24 in N,N-dimethylformamide solution respectively, mix and stir the two under ice bath conditions for 2 h, then add saturated sodium hydroxide ethanol solution to adjust its pH to 8.0, then wash with acetone, dialyze the obtained precipitate in ultrapure water and then freeze-dry to obtain sulfonated hyaluronic acid;

[0057] Dissolve MES, NHS and EDC in ultrapure water in turn to obtain an activator (in the activator, EDC is 1 mg / mL, NHS is 0.24 mg / mL, and MES is 9.76 mg / mL). Add the activator to the sulfonated hyaluronic acid aqueous solution (S-HA, 2 mg / mL) according to the volume ratio of sulfonated hyaluronic acid to activator of 9:1, and quickly add polyethyleneimine solution (PEI, 0.5 mg / mL) with shaking at 37°C to obtain a nanoparticle solution of sulfonated hyaluronic acid; among them, the volume ratio of S-HA to PEI is 9:1; the concentration of the obtained nanoparticle solution of sulfonated hyaluronic acid is 8 mg / mL.

[0058] (3) Stir the Schiff base mixed solution to be sprayed to make it in a relatively stable state, then load it into a syringe, and spray the Schiff base solution onto the surface of the magnesium alloy substrate for vascular stents by electrostatic spraying method. After curing, spray the nanoparticle solution of sulfonated hyaluronic acid onto the surface of the Schiff base coating in the same way. Among them, the voltage of the two electrostatic sprayings is 11 kV, the flow rate is 18 μm / min, and the spraying times are 1.5 min respectively.

[0059] The exclusive coating of the fully degradable magnesium alloy vascular stent material in this example is prepared by the above method.

[0060] Example 2

[0061] This example provides a preparation method for the exclusive coating of the fully degradable magnesium alloy vascular stent material. The magnesium alloy substrate used is specifically ZE21B magnesium alloy.

[0062] Prepare a Schiff base / sulfonated hyaluronic acid nanoparticle coating on the surface of ZE21B magnesium alloy. The specific steps are as follows:

[0063] (1) Dissolve 0.01 mol of methionine in 60 mL of absolute ethanol to obtain an ethanol solution of methionine. Dissolve 0.01 mol of paeonol in 15 mL of absolute ethanol to obtain a paeonol solution. Dropwise add the ethanol solution of methionine into the paeonol solution, reflux at 80 °C for 2 h, then cool, filter, and dry to obtain methionine Schiff base. Then, use glycine and paeonol, lysine and paeonol respectively, and refer to the preparation process and parameters of methionine Schiff base to prepare glycine Schiff base and lysine Schiff base.

[0064] Weigh methionine Schiff base, glycine Schiff base, and lysine Schiff base respectively and dissolve them in absolute ethanol, then stir to make a compound Schiff base mixture solution for standby. In the Schiff base mixture solution, the molar concentrations of methionine Schiff base, glycine Schiff base, and lysine Schiff base are 0.04 M, 0.02 M, and 0.02 M in sequence.

[0065] (2) Fully dissolve sodium hyaluronate with a molecular weight of 2×10 5 Da in ultrapure water (2 mg / mL), add strongly acidic ion exchange resin (the dosage of strongly acidic ion exchange resin corresponding to every 100 mL of sodium hyaluronate solution is 10 g), stir, let it stand, and filter. Then add tetrabutylammonium hydroxide solution (concentration 10%) until it is alkaline, and freeze-dry to obtain quaternary ammonium salt of hyaluronic acid. Dissolve the quaternary ammonium salt of hyaluronic acid and sulfur trioxide pyridine complex with a mass ratio of 1:8 in N,N-dimethylformamide solution respectively, mix and stir them in an ice bath for 2 h, then add saturated sodium hydroxide ethanol solution to adjust the pH to 8.0, and then wash with acetone. Dialyze the precipitate obtained after washing in ultrapure water and then freeze-dry to obtain sulfonated hyaluronic acid;

[0066] Dissolve MES, NHS, and EDC in ultrapure water in sequence to obtain an activator (in the activator, the concentration of EDC is 1 mg / mL, the concentration of NHS is 0.24 mg / mL, and the concentration of MES is 9.76 mg / mL). Add the activator to the sulfonated hyaluronic acid aqueous solution (S-HA, 2 mg / mL) according to the volume ratio of sulfonated hyaluronic acid to activator of 9:1, and quickly add polyethyleneimine solution (PEI, 0.5 mg / mL) with shaking at 37 °C to obtain a nanoparticle solution of sulfonated hyaluronic acid; among them, the volume ratio of S-HA to PEI is 11:1; the concentration of the obtained nanoparticle solution of sulfonated hyaluronic acid is 8 mg / mL.

[0067] (3) Stir the Schiff base mixed solution to be sprayed to make it in a relatively stable state, then load it into a syringe, and spray the Schiff base solution onto the surface of the magnesium alloy substrate for vascular stents by electrostatic spraying. After curing, spray the nanoparticle solution of sulfonated hyaluronic acid onto the surface of the Schiff base coating in the same way. Among them, the electrostatic spraying voltage for both times is 9 kV, the flow rate is 15 μm / min, and the spraying times are 1.5 min respectively.

[0068] The exclusive coating of the fully degradable magnesium alloy vascular stent material in this example is prepared by the above method.

[0069] Example 3

[0070] This example provides a preparation method for the exclusive coating of the fully degradable magnesium alloy vascular stent material. The magnesium alloy substrate used is the Mg-Zn-Y-Nd magnesium alloy.

[0071] Prepare a Schiff base / sulfonated hyaluronic acid nanoparticle coating on the surface of the Mg-Zn-Y-Nd magnesium alloy. The specific steps are as follows:

[0072] (1) Dissolve 0.01 mol of methionine in 60 mL of absolute ethanol to obtain an ethanol solution of methionine, dissolve 0.01 mol of paeonol in 15 mL of absolute ethanol to obtain a paeonol solution, dropwise add the ethanol solution of methionine into the paeonol solution, reflux at 70 °C for 1.5 h, then cool, filter, and dry to obtain methionine Schiff base; then use glycine and paeonol, lysine and paeonol respectively, and refer to the preparation process and parameters of methionine Schiff base to prepare glycine Schiff base and lysine Schiff base.

[0073] Weigh methionine Schiff base, glycine Schiff base, and lysine Schiff base respectively and dissolve them in absolute ethanol, then stir to make a compound Schiff base mixed solution for standby; in the Schiff base mixed solution, the molar concentrations of methionine Schiff base, glycine Schiff base, and lysine Schiff base are 0.04 M, 0.02 M, and 0.02 M in sequence.

[0074] (2) The molecular weight is 1.5×10 5After the sodium hyaluronate of Da was fully dissolved in ultrapure water (2 mg / mL), strong acid type ion exchange resin was added (the dosage of strong acid type ion exchange resin corresponding to every 100 mL of sodium hyaluronate solution was 10 g). After stirring, it was left standing and filtered. Then tetrabutylammonium hydroxide solution (concentration 10%) was added until it was alkaline, and it was freeze-dried to obtain quaternary ammonium salt of hyaluronic acid; the quaternary ammonium salt of hyaluronic acid and sulfur trioxide pyridine complex with a mass ratio of 1:48 were respectively dissolved in N,N-dimethylformamide solution. After the two were mixed and stirred in an ice bath for 2 h, sodium hydroxide saturated ethanol solution was added to adjust its pH to 8.0. Then it was washed with acetone, and the obtained precipitate after washing was dialyzed in ultrapure water and then freeze-dried to obtain sulfonated hyaluronic acid;

[0075] MES, NHS and EDC were successively dissolved in ultrapure water to obtain an activator (in the activator, EDC was 1 mg / mL, NHS was 0.24 mg / mL, and MES was 9.76 mg / mL). According to the volume ratio of sulfonated hyaluronic acid to activator of 9:1, the activator was added to the sulfonated hyaluronic acid aqueous solution (S-HA, 2 mg / mL). Under the condition of 37 °C, polyethyleneimine solution (PEI, 0.5 mg / mL) was added quickly with shaking to obtain a nanoparticle solution of sulfonated hyaluronic acid; among them, the volume ratio of S-HA to PEI was 10:1; the concentration of the obtained nanoparticle solution of sulfonated hyaluronic acid was 8 mg / mL.

[0076] (3) The Schiff base mixed solution to be sprayed was stirred to make it in a relatively stable state, and then loaded into a syringe. The Schiff base solution was sprayed onto the surface of the magnesium alloy substrate for vascular stent by electrostatic spraying method. After curing, the nanoparticle solution of sulfonated hyaluronic acid was sprayed onto the surface of the Schiff base coating in the same way. Among them, the voltage of the two electrostatic sprayings was 10 kV, the flow rate was 21 μm / min, and the spraying times were 1 min respectively.

[0077] The exclusive coating of the fully degradable magnesium alloy vascular stent material in this embodiment was prepared by the above method.

[0078] Test Example 1 Scanning Electron Microscopy and EDS Element Analysis

[0079] Scanning electron microscopy was used to analyze the micro-morphology of the magnesium alloy substrate material sample for vascular stent without coating in Example 1 and the magnesium alloy sample of the exclusive coating of the fully degradable magnesium alloy vascular stent material prepared in Example 1, and EDS element detection and analysis were carried out on different sites (numbered 1-4) in the scanning electron micrograph.

[0080] Figure 2SEM images of the uncoated magnesium alloy substrate material sample for vascular stents used in Example 1 of the present invention (left figure) and the surface of the magnesium alloy sample with a dedicated coating for fully degradable magnesium alloy vascular stent materials prepared (right figure). Table 1 shows Figure 2 the EDS elemental detection and analysis results at different test points in

[0081] Table 1 EDS elemental detection and analysis results

[0082]

[0083] From Figure 2 and the test results in Table 1, it can be seen that nanoparticles with a particle size less than 200 nm are visible on the surface of the magnesium alloy with a dedicated coating for fully degradable magnesium alloy vascular stent materials prepared in Example 1, and significant N and S elements are present, proving that the Schiff base-sulfonated hyaluronic acid nanoparticle composite coating has been successfully prepared on the surface of the magnesium alloy substrate.

[0084] Test Example 2 Degradation inhibition and anticoagulant performance tests

[0085] Figure 3 are the corrosion morphologies and blood component adhesion results after soaking in human blood for 7 days of the uncoated magnesium alloy substrate material sample for vascular stents in Example 1 (left figure) and the magnesium alloy sample with a dedicated coating for fully degradable magnesium alloy vascular stent materials prepared in Example 1 (right figure).

[0086] From Figure 3 it can be seen that the surface degradation products and blood components of the Schiff base / sulfonated hyaluronic acid nanoparticles in Example 1 are significantly less than those on the surface of the uncoated magnesium alloy, proving that the composite coating of the present invention can endow the magnesium alloy with better magnesium alloy degradation inhibition function and anticoagulant function.

[0087] Test Example 3 Fluorescent staining of endothelial cells and light microscopy and migration distance analysis

[0088] Figure 4 are the fluorescent staining results of endothelial cells on the surface of the uncoated magnesium alloy substrate material sample for vascular stents in Example 1 (left figure) and the magnesium alloy sample with a dedicated coating for fully degradable magnesium alloy vascular stent materials prepared in Example 1 (right figure).

[0089] From Figure 4 it can be seen that the number of endothelial cells on the surface of the Schiff base / sulfonated hyaluronic acid nanoparticle coating in Example 1 is significantly more than that on the surface of the uncoated magnesium alloy.

[0090] Figure 5Light microscopy images (Figure a) of vascular endothelial cells migrating on the magnesium alloy substrate material sample without coating in Example 1 and the magnesium alloy sample with the exclusive coating for fully degradable magnesium alloy vascular stent materials prepared in Example 1, as well as the statistical results of the migration distance (Figure b).

[0091] It can be seen from Figure 5 that the migration distance of endothelial cells on the surface of the Schiff base / S-HA nanoparticle coating in Example 1 is significantly higher than that of the magnesium alloy substrate without coating, indicating that the coating of the present invention can effectively improve the endothelialization function.

[0092] Test Example 4 Smooth muscle cell culture and fluorescence staining

[0093] Contractile smooth muscle cells are normal human cells, which are beneficial to the endothelialization of the material surface and can reverse the hyperplastic pathological phenomenon. Figure 6 This shows the results of smooth muscle cell culture on the magnesium alloy substrate for vascular stents without coating and the magnesium alloy with the exclusive coating for fully degradable magnesium alloy vascular stent materials in Example 1. Among them Figure 6 (a) is the fluorescence staining image of smooth muscle cells (green is the staining of the specific factor α-SMA of normal human contractile smooth muscle cells, and blue is the nuclear staining); Figure 6 (b) is the three-dimensional imaging map of the fluorescence intensity Ipwin32 of α-SMA.

[0094] It can be seen from Figure 6 that the expression of the α-SMA factor in smooth muscle cells on the surface of the Schiff base / S-HA nanoparticle coating in Example 1 is significantly higher than that on the magnesium alloy surface without coating modification, proving that the coating of the present invention can endow the magnesium alloy with better anti-proliferation function and endothelialization promotion function.

[0095] Test Example 5 Macrophage culture and anti-inflammatory test

[0096] M1 macrophages are inflammatory cells, while M2 macrophages are anti-inflammatory immune cells. M2 macrophages are beneficial to the endothelialization of the material surface and can inhibit the inflammatory pathological phenomenon. Figure 7 This shows the results of macrophage culture on the magnesium alloy substrate for vascular stents without coating and the magnesium alloy with the exclusive coating for fully degradable magnesium alloy vascular stent materials in Example 1. Among them Figure 7 (a) is the fluorescence staining image of macrophages (in the color picture, green is the staining of the specific factor CD206 of M2 macrophages, red is the staining of the specific factor TNF-α of M1 macrophages, and blue is the nuclear staining); Figure 7 (b) is the three-dimensional imaging map of the fluorescence intensity Ipwin32 of TNF-α and CD206.

[0097] It can be seen from Figure 7It can be seen that the expression of macrophage TNF-α factor on the surface of the Schiff base / S-HA nanoparticle coating in Example 1 is significantly lower than that on the surface of the bare magnesium alloy, and the expression of CD206 factor is significantly higher than that on the surface of the magnesium alloy without coating modification, which proves that the coating of the present invention can endow the magnesium alloy with better anti-inflammatory function and endothelialization function.

[0098] Test Example 6 Anticoagulation Test

[0099] Figure 8 This is a graph showing the morphology and quantity of platelets adhered to the surface of the magnesium alloy substrate sample without coating for the vascular stent in Example 1 of the present invention (left figure) and the Schiff base-S-HA nanoparticle-coated magnesium alloy sample prepared in Example 1 (right figure) after being immersed in human blood rich in platelet plasma for 1 hour.

[0100] It can be seen from Figure 8 that the number of platelets on the surface of the Schiff base-S-HA nanoparticle-coated magnesium alloy sample is significantly less than that of the sample without coating, which proves that the Schiff base-S-HA nanoparticle coating can inhibit the adhesion of platelets on the surface of the magnesium alloy. Platelet adhesion is an important index for blood coagulation. Therefore, this result proves that the Schiff base-S-HA nanoparticle coating can significantly improve the anticoagulation function of the magnesium alloy surface.

[0101] In summary, the present invention uses electrostatic spraying to deposit a compound Schiff base on the surface of the magnesium alloy to form a dense coating with a degradation inhibition function, and then grafts and synthesizes sulfonated hyaluronic acid nanoparticles on the surface of the Schiff base coating by electrostatic spraying to form a Schiff base-sulfonated hyaluronic acid nanoparticle functional active composite coating, which can reduce the degradation rate of the magnesium alloy and endow the magnesium alloy stent material with comprehensive functions of anticoagulation, anti-inflammation, endothelialization, anti-proliferation and intracellular delivery of magnesium ions. Based on this, the multifunctional active coating constructed on the surface of the magnesium alloy material in the present invention can significantly improve the service life and quality of the magnesium alloy stent, and has good application prospects in the fields of coatings and modification materials for magnesium alloy stents.

Claims

1. A preparation method of an exclusive coating for a fully degradable magnesium alloy vascular stent material, characterized in that It includes the following steps: (1) Dissolve methionine Schiff base, glycine Schiff base and lysine Schiff base in an organic solvent to obtain a Schiff base mixture solution; React sodium hyaluronate with a quaternary ammonium salt to obtain a quaternary ammonium salt of hyaluronic acid; then carry out a substitution reaction between the quaternary ammonium salt of hyaluronic acid and a sulfur trioxide pyridine complex to obtain sulfonated hyaluronic acid; in the presence of an activator, react sulfonated hyaluronic acid with polyethyleneimine to obtain a nanoparticle solution of sulfonated hyaluronic acid; Among them, the molecular weight of the sodium hyaluronate is 1×10 5 ~2×10 5 Da; the quaternary ammonium salt is tetrabutylammonium hydroxide; when the quaternary ammonium salt of hyaluronic acid reacts with the pyridine sulfur trioxide complex by substitution reaction, the mass ratio of the quaternary ammonium salt of hyaluronic acid to the pyridine sulfur trioxide complex is 1:(8-48); (2) Adopt the electrostatic spraying method to first spray the Schiff base mixture solution on the surface of the magnesium alloy substrate, and then spray the nanoparticle solution of sulfonated hyaluronic acid after curing, thus obtaining a special coating for the fully degradable magnesium alloy vascular stent material; the voltage of the electrostatic spraying is 9-11 kV, the spraying flow rate is 15-21 μm / min, and the spraying time is 1.0-1.5 min.

2. The preparation method of the exclusive coating for the fully degradable magnesium alloy vascular stent material according to claim 1, wherein, In step (1), the methionine Schiff base is prepared by carrying out a thermal reflux reaction between methionine and paeonol; The glycine Schiff base is prepared by carrying out a thermal reflux reaction between glycine and paeonol; The lysine Schiff base is prepared by carrying out a thermal reflux reaction between lysine and paeonol.

3. The preparation method of the exclusive coating for the fully degradable magnesium alloy vascular stent material according to claim 2, characterized in that, The temperature of the thermal reflux reaction is 60-80 °C, and the time of the thermal reflux reaction is 1.5-3 h.

4. The preparation method of the exclusive coating for the fully degradable magnesium alloy vascular stent material according to claim 1, characterized in that, In step (1), the organic solvent is absolute ethanol; in the Schiff base mixture solution, the molar concentrations of methionine Schiff base, glycine Schiff base and lysine Schiff base are 0.04 M, 0.02 M and 0.02 M in sequence.

5. The preparation method of the exclusive coating for the fully degradable magnesium alloy vascular stent material according to claim 1, wherein, In step (1), the activator is a ternary combination of sodium fatty acid methyl ester sulfonate, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide.

6. The preparation method of the exclusive coating for the fully degradable magnesium alloy vascular stent material according to claim 1, characterized in that, In step (1), the reaction between sulfonated hyaluronic acid and polyethyleneimine is carried out as follows: prepare an aqueous solution of sulfonated hyaluronic acid with a concentration of 2 mg / mL, and then add an aqueous solution of polyethyleneimine with a concentration of 0.5 mg / mL under shaking at 25-40 °C to obtain a nanoparticle solution of sulfonated hyaluronic acid.

7. The preparation method of the exclusive coating for the fully degradable magnesium alloy vascular stent material according to claim 6, characterized in that, The volume ratio of the aqueous solution of sulfonated hyaluronic acid to the aqueous solution of polyethyleneimine is (9-11):

1.

8. A special coating for a fully degradable magnesium alloy vascular stent material prepared by the preparation method according to any one of claims 1-7.

9. The application of the exclusive coating for the fully degradable magnesium alloy vascular stent material according to claim 8, wherein, Application in the preparation of a functional coating for a fully degradable magnesium alloy vascular stent.

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