CN115607728A - Long-term catalytic nitric oxide release and anticoagulation coating and preparation method thereof - Google Patents
Long-term catalytic nitric oxide release and anticoagulation coating and preparation method thereof Download PDFInfo
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- A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/42—Anti-thrombotic agents, anticoagulants, anti-platelet agents
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- A61L2420/00—Materials or methods for coatings medical devices
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
Abstract
The invention discloses a long-term catalytic nitric oxide release and anticoagulation coating and a preparation method thereof 2 The preparation method of the coating comprises the following steps of: takes a metal-based biomaterial as an anode, and adopts an electrochemical oxidation method to prepare regular TiO on the surface of the metal-based biomaterial 2 Nanotube layer, trunkAnd after drying and annealing treatment, carrying out hydrothermal treatment in a solution containing metal ions, and then sequentially placing the solution in a mixed solution of dopamine solution and lysine/heparin/selenocysteine for deposition reaction to obtain the coating. The coating prepared by the invention has excellent anticoagulation property, biocompatibility and bioactivity, can realize the long-term release of catalytic nitric oxide, promotes the endothelialization of the metal-based biological material, ensures the functional expression of normal endothelial cells, is simple and easy to implement in the preparation method, and has good stability.
Description
Technical Field
The invention relates to a bio-based coating and a preparation method thereof, in particular to a long-term nitric oxide catalytically-releasing and anticoagulation coating and a preparation method thereof.
Background
The titanium alloy has excellent mechanical property, corrosion resistance and biocompatibility, so that the titanium alloy is applied to human tissue implantation substitution materials of blood, orthopedics, teeth and the like. Among blood contact materials, titanium and its alloys are often used as materials for heart valves, vascular stents, blood circulation aids, and the like. However, the surfaces of titanium and titanium alloys are biologically inert, lack anticoagulant activity, and do not induce the formation of the natural anticoagulant layer, the endothelial cell layer. When the material is used as a long-term implant material, a large amount of blood platelets and plasma proteins are adhered to the surface of the implant material, so that reactions such as thrombus, vascular restenosis, inflammation and the like are caused, and the surface of the implant material is weaker in the binding capacity with human tissues, so that the implant failure is caused. In addition, titanium alloys as blood contact materials require rapid endothelialization, and it is desired that the surface of titanium materials can promote the functional expression of normal endothelial cells.
Healthy endothelial cells maintain vascular tone, inhibit platelet aggregation and activation, and inhibit proliferation and migration of smooth muscle cells by synthesizing nitric oxide vasodilator factor. At present, the nitric oxide signal molecule is introduced to the surface of the biological material, exogenous nitric oxide donor molecules can be fixed on the surface of the biological material, and the signal molecule is slowly released after the nitric oxide signal molecule is implanted into the body. However, the surface-mounted nitric oxide donors are very limited, and cannot achieve the purpose of releasing nitric oxide for a long time. Human blood contains a large number of endogenous nitric oxide carriers. The catalytic molecules released by nitric oxide molecules are fixed on the surface of the titanium material, so that the release of endogenous nitric oxide in human plasma can be catalyzed.
The invention patent with publication number CN102950102 discloses a method for preparing a multi-growth factor slow release coating on the surface of titanium and titanium alloy, which takes nano particles as growth factor carriers and takes a charged polymer film as a medium to prepare a multi-layer coating containing the nano particles of the growth factors on the surface of the titanium. The nano-particles contained in the coating are prepared by mixing a polycation solution containing dopamine with a certain concentration and a polyanion solution containing growth factors. The nano-particles have good dispersibility, can protect the activity of growth factors, and are easy to fix on the surface of a material. By controlling the electrical property of the nano particles and the polymer films, the nano particles loaded with different factors are embedded between the polymer films. By adjusting the loading amount, the loading sequence and the coating thickness of different nanoparticles, the release rate, the release amount and the release sequence of various growth factors can be effectively controlled. However, the preparation method of the patent is only limited to the preparation of bone tissue repair and bone material promotion, and the carried growth factors are also growth factors such as BMP2, TGF-beta, FGF, IGF and the like which promote bone formation, and no mention is made as to whether the preparation of the cardiovascular material is beneficial or not.
The invention patent with publication number CN108969805 discloses an anticoagulant hydrogel coating capable of catalyzing nitric oxide release and a preparation method thereof, and the patent comprises the following steps: (1) pretreating a substrate material; (2) At room temperature, sequentially placing the pretreated substrate material in a polycation electrolyte, a mixed solution of polyphenol and a compound A and a polycation electrolyte for reaction for 1-20min, and then cleaning for 3-5 times; (3) And (3) at room temperature, repeating the operation of the step (2) for 5-20 times by using the product obtained in the step (2) as a substrate, and drying by nitrogen to obtain the anticoagulant hydrogel coating capable of catalyzing the release of nitric oxide. The preparation of the patent adopts an electrostatic adsorption mode, and has certain influence on the long-term stability of the coating. In addition, the electrostatically adsorbed catalysts are significantly limited in number.
Disclosure of Invention
The purpose of the invention is as follows: in order to solve the technical problems in the prior art, the invention aims to provide a coating which has excellent anticoagulation activity and bioactivity and can realize long-term catalytic release of nitric oxide molecules, and also provides a preparation method of the coating.
The technical scheme is as follows: the long-term catalytic nitric oxide release and anticoagulation coating takes a metal-based biomaterial as a base layer and comprises anatase TiO 2 A nanotube layer, a metal ion layer, a dopamine layer and a lysine/heparin/selenocysteine combined layer, wherein the metal ion layer is carried on anatase TiO 2 The nanotube layer is combined with dopamine layer, and the other side of dopamine layer is combined with lysine/heparin/selenocysteine combined layer.
Further, the anatase type TiO 2 The inner diameter of the nanotube is 50-120nm, the length of the nanotube is 1-5 μm, and the metal ions are one or two of copper ions or zinc ions.
Further, the metal-based biomaterial is a titanium alloy, preferably one of TA1 alloy, TA2 alloy, or TC4 alloy.
The preparation method of the anti-coagulation coating capable of catalytically releasing nitric oxide for a long time comprises the following steps:
(1) Takes a metal-based biomaterial as an anode, and adopts an electrochemical oxidation method to prepare regular TiO on the surface of the metal-based biomaterial 2 Drying the nanotube layer, and then annealing to obtain anatase;
(2) Carrying out hydrothermal treatment on the material prepared in the step (1) in a solution containing metal ions;
(3) Placing the material prepared in the step (2) in a dopamine solution for deposition;
(4) Dissolving lysine in Tris buffer solution, adding heparin, fully stirring for reaction, then adding an activating agent for activation, adding selenocysteine, and reacting to obtain a lysine/heparin/selenocysteine mixed solution;
(5) And (4) soaking the material prepared in the step (3) in the lysine/heparin/selenocysteine mixed solution prepared in the step (4), carrying out oscillation reaction, washing with water and drying to obtain the anticoagulant coating capable of catalytically releasing nitric oxide for a long time.
Further, in the step (1), the conditions of the electrochemical oxidation are as follows: a constant voltage mode, an oxidation voltage of 30-100V, an oxidation time of 30-120min, an organic electrolyte of ethylene glycol +0.5wt% ammonium fluoride +2vol.% deionized water, 0.5wt% HF or ethylene glycol +1.5wt% ammonium fluoride +5vol.% deionized water, an annealing temperature of 400-500 ℃, and an annealing time of 1-5h.
Further, in the step (2), the concentration of the solution containing the metal ions is 0.5-2.5mol/L, the temperature of the hydrothermal treatment is 150-200 ℃, and the time of the hydrothermal treatment is 1-5h.
Further, in the step (3), the pH value of the dopamine solution is 8.0-8.5, and the soaking time is 10-24h.
Further, in the step (4), the mass ratio of lysine to heparin is 5:3-5:2, the concentration of lysine is 0.5-5mg/ml, preferably 2.5mg/ml, and the conditions for fully stirring and reacting lysine and heparin are as follows: reacting for 1-5h in an environment at 2-8 ℃, wherein the activating agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide, the molar ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to the N-hydroxysuccinimide is 3, the concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is 1-3mg/ml, preferably 2.5mg/ml, the activation time is 10-30min, the concentration of the selenocysteamine is 0.5-1.5mmol/L, and the shaking reaction time is 5-10h.
The invention principle is as follows: the invention utilizes TiO prepared by anodic oxidation annealing treatment 2 The inner diameter of the nanotube array is 50-120nm, and the length of the nanotube array is 1-5 μm. Doping Cu in the nanotube by hydrothermal method 2+ 、Zn 2+ One or more of (a). It has been shown that both metal ions promote the functional expression of normal endothelial cells, with Cu 2+ Has obvious catalytic action on the release of endogenous NO molecules in blood plasma. The nanotube array modified by dopamine and carrying metal ions can play a role of a middle layer for the fixation of anticoagulant molecules and NO catalytic release molecules. Heparin has strong anticoagulant property and is a clinically common anticoagulant and thrombus inhibiting medicament. Selenocysteine is a small molecular compound containing selenium and has an important function of catalyzing endogenous NO release. The characteristics that lysine has positive charges in a solution and heparin has negative charges are utilized, the lysine and the heparin are assembled by static electricity, and then carboxyl of the lysine and amino of selenocysteine are subjected to amide reaction and are covalently combined, so that the heparin and the selenocysteine are assembled together; and then the oxidized dopamine is combined on the surface of the titanium alloy through Schiff base reaction between a large number of amino groups in lysine and selenocysteine and quinone groups of the oxidized dopamine.
Has the advantages that: compared with the prior art, the invention has the following remarkable advantages:
(1) According to the invention, the regular nanotube array is prepared on the surface of the titanium alloy, so that the surface activity and the hydrophilicity of the titanium material are increased, and good sites are provided for subsequent carrying of ions and molecules; the heparin molecules have a good anticoagulation function, and the carried metal ions and selenocysteine molecules have excellent capacity of catalyzing the release of endogenous NO donors, so that the heparin molecules can realize various physiological functions as implant materials and can be applied to blood contact materials;
(2) According to the invention, through electrostatic self-assembly and covalent combination, heparin and selenocysteine molecules can be simultaneously fixed on the surface of the titanium alloy;
(3) The preparation technology of the invention has the advantages of mild conditions, no need of special equipment, simple process and low cost.
Drawings
FIG. 1 is an electron micrograph of a coating obtained in example 2 of the present invention, wherein (a) and (c) are TiO treated by anodizing and annealing the surface of a titanium alloy substrate 2 Nano-meterThe tube layers, (b) and (d) are metal ion-loaded active coatings with catalytic NO release;
FIG. 2 is a water contact angle profile of a coating produced in example 2 of the present invention, (a) is a water contact angle formed by a titanium alloy substrate: 51.2 ° ± 0.07 °, (b) is the water contact angle of the active coating with supported metal ions which takes titanium alloy as the base layer and catalyzes NO release: 13.2 ° ± 0.02 °;
FIG. 3 is a graph showing the number of adhered platelets on the surface of the titanium alloy substrate and the coating layer obtained in example 2 of the present invention;
FIG. 4 shows the VEGF expression of cells cultured on the surface of Ti alloy substrate and coating prepared in example 2 of the present invention for 1 day and 3 days;
FIG. 5 shows the NO release amount of cells cultured on the surface of the titanium alloy substrate and the titanium alloy coating prepared in example 2 of the present invention after 1 day and 3 days.
Detailed Description
The present invention will be further described with reference to the following specific embodiments and the accompanying drawings.
Example 1: the long-term catalytic nitric oxide release and anticoagulation coating provided by the invention takes TA1 alloy as a base layer and comprises anatase TiO 2 A nanotube layer, a copper ion/zinc ion layer, a dopamine layer and a lysine/heparin/selenocysteine combined layer.
The preparation method of the coating comprises the following steps: preparing TA1 alloy into a sample of 50mm × 50mm × 2.5mm, polishing the surface with No. 320, 400, 600, 800 and 1000 abrasive paper, and polishing with HF + HNO 3 After the mixed solution is subjected to electrolytic polishing for 5s, washing and drying; performing anodic oxidation, oxidizing in an electrolyte of ethylene glycol, 0.5wt% of ammonium fluoride and 2vol.% of deionized water, maintaining the voltage at 60V, oxidizing for 30min, ultrasonically cleaning water, ethylene glycol and ethanol for 5min respectively, and drying; then, the sample is put into a reaction kettle, and ZnCl is added 2 、CuCl 2 Respectively 0.5M, reacting at 200 ℃ for 2h, taking out, washing with water for 3 times, and air-drying; a1 mg/ml dopamine solution was prepared with Tris buffer and the pH of the solution was adjusted to 8.0. The prepared TiO carrying the metal ions 2 Placing the nanotube into the dopamine solution, reacting for 12 hours at room temperature, washing with water, and drying; 2.5 mg/mlL-lysine and 1.5mg/ml heparinAdding sodium into a Tris solution, stirring, placing in an environment with the temperature of 2-8 ℃ for 10h, taking out, adding 2.5mg/ml of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 12mg/ml of N-hydroxysuccinimide for activation for 20min, adding 1mM of selenocysteine to prepare a mixed solution, then placing the regular nanotube array carrying metal ions into the mixed solution, and fully reacting for 8h to obtain the metal ion-carrying nano-tube array.
Example 2: the long-term nitric oxide releasing and anticoagulation coating provided by the invention takes TC4 alloy as a base layer and comprises anatase TiO 2 A nanotube layer, a copper ion layer, a dopamine layer and a lysine/heparin/selenocysteine combined layer.
The preparation method of the coating comprises the following steps: preparing TC4 alloy into 10mm × 10mm × 2.5mm sample, grinding the surface with 320, 400, 600, 800, 1000# sand paper, polishing, HF + HNO 3 The mixed solution is washed by water and dried after being electropolished for 3 s; anodizing in 0.5wt% HF electrolyte, maintaining the voltage at 45V, oxidizing for 1h, ultrasonically cleaning with water, ethylene glycol and ethanol for 5min, and drying; subsequently, the sample was placed in a reaction kettle and 0.5M CuCl was added 2 Reacting at 200 ℃ for 2h, taking out, washing with water for 3 times, and air-drying; a1 mg/ml dopamine solution was prepared with Tris buffer and the pH of the solution was adjusted to 8.5. Placing the prepared TiO2 nanotube carrying the metal ions into the dopamine solution, reacting for 12 hours at room temperature, washing with water, and drying; adding 2.5 mg/mlL-lysine and 1.5mg/ml heparin sodium into a Tris solution, stirring, placing in an environment at 2-8 ℃ for 10h, taking out, adding 2.5mg/ml 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 12mg/ml N-hydroxysuccinimide for activation for 20min, and adding 1mM selenocysteine to prepare a mixed solution. And then placing the regular nanotube array carrying the metal ions into the mixed solution, and fully reacting for 8 hours to obtain the metal ion-carrying nanotube array.
Example 3: the long-term catalytic nitric oxide release and anticoagulation coating takes TA2 medical alloy as a base layer and comprises anatase TiO 2 A nanotube layer, a copper ion/zinc ion layer, a dopamine layer and a lysine/heparin/selenocysteine combined layer.
The preparation method of the coating comprises the following steps: made of TA2 medical titanium alloy50mm × 50mm × 2.5mm sample, surface polished with No. 320, 400, 600, 800, 1000 sand paper, and HF + HNO 3 After the mixed solution is subjected to electrolytic polishing for 5s, washing and drying; performing anodic oxidation, oxidizing in electrolyte of ethylene glycol, 1.5wt% of ammonium fluoride and 5vol.% of deionized water, maintaining the voltage at 100V, oxidizing for 1h, ultrasonically cleaning water, ethylene glycol and ethanol for 5min respectively, and drying; then, the sample is put into a reaction kettle, and ZnCl is added 2 、CuCl 2 Respectively 0.5M, reacting at 180 ℃ for 3h, taking out, washing with water for 3 times, and air-drying; a1 mg/ml dopamine solution was prepared with Tris buffer and the pH of the solution was adjusted to 8.5. Placing the prepared TiO2 nanotube carrying the metal ions into the dopamine solution, reacting for 12 hours at room temperature, washing with water, and drying; adding 2.5 mg/mlL-lysine and 1mg/ml heparin sodium into a Tris solution, stirring, placing in an environment at 2-8 ℃ for 4h, taking out, adding 2.5mg/ml 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 12mg/ml N-hydroxysuccinimide for activation for 20min, and adding 0.5mM selenocysteine to prepare a mixed solution. And then placing the regular nanotube array carrying the metal ions into the mixed solution, and fully reacting for 8 hours to obtain the metal ion-carrying nanotube array.
Fig. 1 to 5 show the data obtained in example 2 concerning the characteristics of nanotube coatings, platelet adhesion and the expression of endothelial cell function. The nanotubes prepared using a voltage of 45V had diameters of about 60-80nm and tube lengths of about 3 μm. After carrying metal ions, depositing dopamine and fixing the lysine/heparin/selenocysteine combined layer, the diameter of the nanotube is obviously reduced. Because a large number of hydrophilic groups (such as-OH, -COOH and the like) are introduced into the surface of the titanium alloy, the water contact of the coating is obviously reduced greatly compared with the titanium alloy. The number of platelet adhesion at the coated surface is significantly reduced. Endothelial cells are respectively inoculated on the surfaces of the titanium alloy and the coating and cultured for 1 and 3 days, and the concentration of VEGF and NO which are detected to be expressed by cell functions is obviously improved.
Claims (10)
1. A long-term catalytic nitric oxide release and anticoagulant coating based on a metal-based biomaterial, characterized in that the coating comprises anatase TiO 2 Nanotube layer, metal ion layer, dopamine layer and lysine/heparin/selenocysteineA combined layer, the metal ion layer being carried on anatase type TiO 2 And the other side of the dopamine layer is combined with a lysine/heparin/selenocysteine combined layer.
2. Coating according to claim 1, characterized in that the anatase TiO is 2 The inner diameter of the nanotube is 50-120nm, and the length of the nanotube is 1-5 μm.
3. The coating of claim 1, wherein the metal ions are one or both of copper ions and zinc ions.
4. A method for preparing a long-term catalytic nitric oxide releasing and anticoagulant coating according to claim 1, comprising the steps of:
(1) Takes a metal-based biomaterial as an anode, and adopts an electrochemical oxidation method to prepare regular TiO on the surface of the metal-based biomaterial 2 Drying the nanotube layer, and then annealing to obtain anatase;
(2) Carrying out hydrothermal treatment on the material prepared in the step (1) in a solution containing metal ions;
(3) Placing the material prepared in the step (2) in a dopamine solution for deposition reaction;
(4) Dissolving lysine in Tris buffer solution, adding heparin, fully stirring for reaction, then adding an activating agent for activation, adding selenocysteine, and reacting to obtain a lysine/heparin/selenocysteine mixed solution;
(5) And (4) placing the material prepared in the step (3) in the lysine/heparin/selenocysteine mixed solution prepared in the step (4), carrying out oscillation reaction, washing with water and drying to obtain the anticoagulant coating capable of catalytically releasing nitric oxide for a long time.
5. The method according to claim 4, wherein in the step (1), the conditions of the electrochemical oxidation are as follows: the oxidation voltage is 30-100V, the oxidation time is 30-120min, the annealing temperature is 400-500 ℃, and the annealing time is 1-5h.
6. The preparation method according to claim 4, wherein in the step (2), the concentration of the solution containing metal ions is 0.5-2.5mol/L, the temperature of the hydrothermal treatment is 150-200 ℃, and the time of the hydrothermal treatment is 1-5h.
7. The method according to claim 4, wherein in the step (4), the mass ratio of lysine to heparin is 5:3-5:2, and the concentration of lysine is 0.5-5mg/ml.
8. The method according to claim 4, wherein in the step (4), the reaction conditions of lysine and heparin are as follows: and (3) performing reaction for 1-5h at the temperature of 2-8 ℃.
9. The method according to claim 4, wherein in the step (4), the activating agents are 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide, and the activating time is 10-30min.
10. The method according to claim 4, wherein in the step (4), the concentration of the selenocysteine is 0.5-1.5mmol/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211266044.XA CN115607728B (en) | 2022-10-17 | 2022-10-17 | Coating capable of releasing nitric oxide and resisting coagulation for long-term catalysis and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211266044.XA CN115607728B (en) | 2022-10-17 | 2022-10-17 | Coating capable of releasing nitric oxide and resisting coagulation for long-term catalysis and preparation method thereof |
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