A thickness-controllable phosphorylcholine monomolecular coating and preparation method thereof

Abstract

The present invention provides a monomolecular phosphorylcholine coating with controllable thickness and a preparation method thereof. The preparation method extends the length of the molecule by covalent assembly layer by layer through Michael addition reaction, thereby controlling the length of the molecular chain and further controlling the thickness of the coating. The raw materials used are inexpensive, which can significantly reduce the product cost. The present invention also provides a monomolecular phosphorylcholine coating with controllable thickness prepared by the preparation method as described above, and an in vivo interventional surgery medical device comprising the coating.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and in particular relates to a thickness-controllable phosphorylcholine monomolecular coating and a preparation method thereof. Background Art

[0002] Blood-contact medical devices have been widely used in clinical practice, such as intravenous catheters, coronary stents, artificial heart valves, hemodialysis circulation devices, membrane oxygenators and other medical devices. When these medical devices come into contact with blood, they will stimulate the host's blood defense mechanism, causing thrombosis and embolism, endangering the patient's life. Therefore, it is of great significance to improve the blood compatibility of biomedical materials and enhance the anticoagulant properties of the materials.

[0003] Heart stents, also known as coronary artery stents, are commonly used medical devices in cardiac interventional surgery and have the function of clearing arterial blood vessels. The main materials are stainless steel, nickel-titanium alloy or cobalt-chromium alloy. When the heart stent material comes into contact with blood, coagulation reaction and thrombus formation will occur, so it must be surface modified to improve its biocompatibility to avoid thrombosis. In the production cost structure of heart stents, although the skeleton metal material accounts for the largest material weight, it accounts for the smallest proportion of cost, while the coating material has a higher technical content and is more expensive. Therefore, the cost of a heart stent mainly comes from the proportion of coating costs. China consumes about 100 tons of coating materials for heart stents each year. Therefore, the development of anti-coagulant coatings has broad market prospects.

[0004] Phosphocholine groups are widely present in the outer layer of biological membranes. The presence of a quaternary ammonium group and a phosphate group in its structure allows it to maintain equal positive and negative charges in the same molecule. This group with equal positive and negative charges can bind a large number of water molecules to form a hydration layer on the surface, thereby reducing the adsorption of proteins and platelets, and therefore has excellent anticoagulant properties. Since phosphorylcholine has both excellent biocompatibility and anticoagulant properties, the introduction of molecules or polymers containing phosphorylcholine groups into the surface of medical devices by physical or chemical methods to obtain anticoagulant properties has been widely studied and applied. Patent CN201480063475.3 discloses a medical device with a phosphorylcholine outer surface, and 2-methacryloyloxyethyl phosphorylcholine (MPC) or its derivatives are fixed on the surface of the stent using a silane coupling agent. CN200510050675.8 discloses a method for improving the biocompatibility of a biomedical stainless steel device, and (MPC) is fixed on the stainless steel surface using a silane coupling agent.

[0005] It can be seen from the prior art that the main problems with the current phosphorylcholine coating are: (1) the coating thickness is uncontrollable; and (2) the phosphorylcholine used is mostly sourced from 2-methacryloyloxyethyl phosphorylcholine (MPC) or its derivatives, which are expensive and lead to high costs. Summary of the invention

[0006] In view of the above problems, the object of the present invention is to provide a monomolecular phosphorylcholine coating with controllable thickness and a preparation method thereof.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing a monomolecular coating of phosphorylcholine with controllable thickness, wherein the preparation method extends the length of the molecule layer by layer through Michael addition reaction, and comprises the following steps:

[0009] Step (1), hydroxylating the surface of the substrate material;

[0010] Step (2), aminate the surface obtained in step (1);

[0011] Step (3), grafting double bond functional groups onto the surface obtained in step (2);

[0012] Step (4), grafting amino functional groups onto the surface obtained in step (3);

[0013] Step (5), grafting phosphorylcholine groups onto the surface obtained in step (4);

[0014] Step (6), adding a reducing agent to the surface obtained in step (5) to reduce the imine bond;

[0015] Wherein, step (3) and step (4) can be repeated for multiple times to control the coating thickness, thereby obtaining a monomolecular coating with a controllable thickness and containing a phosphorylcholine group at the end.

[0016] Preferably, the coating has a thickness of 1-10 nm.

[0017] Preferably, in step (1), the method for hydroxylating the surface of the substrate material comprises: immersing the substrate material in piranha washing solution and stirring for 5-30 minutes at a temperature of 20-60° C., and drying with nitrogen after washing.

[0018] Preferably, in step (2), the surface amination comprises: immersing the substrate in toluene, adding 3-aminopropyltriethoxysilane to prepare a solution with a volume fraction of 1%-10%; stirring and refluxing at 20-80° C. in a nitrogen atmosphere for 4-24 hours, ultrasonically cleaning with ethanol and deionized water, and vacuum drying to obtain an amino-functionalized substrate surface.

[0019] Preferably, in step (3), the surface grafting double bond functional groups comprises: dissolving a diene in tetrahydrofuran to prepare a solution with a concentration of 1-100 mmol / L, immersing the amination-treated substrate in the tetrahydrofuran solution, and stirring the reaction in a nitrogen atmosphere for 4-24 hours; preferably, the diene is selected from one or more of 1,3-butadiene, 1,4-pentadiene, isoprene, and 1,5-hexadiene.

[0020] Preferably, in step (4), the grafting of amino functional groups comprises: dissolving a diamino compound in tetrahydrofuran to prepare a solution with a concentration of 1-100 mmol / L, immersing the substrate grafted with double bond functional groups in the tetrahydrofuran solution, and stirring the reaction in a nitrogen atmosphere for 4-24 hours; preferably, the diamino compound is selected from one or more of ethylenediamine, 1,3-propylenediamine, and 1,4-butylenediamine.

[0021] Preferably, in step (5), the surface grafting of phosphorylcholine groups comprises: dissolving aldehyde phosphorylcholine in anhydrous methanol to prepare a solution with a mass fraction of 1%-10%; immersing the grafted amino-functional substrate in the methanol solution and stirring the reaction in a nitrogen atmosphere for 4-24 hours.

[0022] Preferably, in step (6), reducing the imine bond comprises: dissolving sodium borohydride in anhydrous methanol to prepare a solution with a concentration of 1-100 mmol / L. Immersing the substrate grafted with phosphorylcholine groups in the methanol solution and stirring the reaction for 4-24 hours.

[0023] In a second aspect, the present invention also provides a thickness-controllable phosphorylcholine monomolecular coating prepared by the preparation method described above.

[0024] In a third aspect, the present invention also provides an in vivo interventional surgical medical device, comprising the thickness-controllable phosphorylcholine monomolecular coating as described above.

[0025] In the present invention, the surface of the heart stent is modified by using the above-mentioned monomolecular phosphorylcholine coating with controllable thickness to improve its biocompatibility and avoid the formation of thrombus.

[0026] The present invention provides a method for preparing a monomolecular coating containing phosphorylcholine groups with controllable thickness. The preparation method firstly hydroxylates the surface of a substrate by an acid washing method, then grafts a silane coupling agent, and then controls the molecular chain extension by cyclic reaction layer by layer assembly using butadiene and propylenediamine through Michael addition reaction, and the thickness can be increased by 1 to 2 nm per cycle, and finally grafts phosphorylcholine groups through Schiff base reaction. The core of the present invention is to design the cyclic reaction layer by layer assembly of butadiene and propylenediamine to control the length of the molecular chain, thereby controlling the coating thickness.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention controls the length of the molecular chain and further controls the thickness of the coating by designing a repeated cycle of Michael addition reaction layer by layer covalent assembly;

[0029] (2) The raw materials used are cheap, which can significantly reduce product costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the molecular formula of the coating prepared by the present invention, wherein n is the number of cycles of step (3) and step (4);

[0031] Figure 2 The molecular formula of the coating prepared in Example 1 of the present invention;

[0032] Figure 3 The molecular formula of the coating prepared in Example 2 of the present invention;

[0033] Figure 4 The molecular formula of the coating prepared in Example 3 of the present invention;

[0034] Figure 5 (ad) are the adhesion of platelets on the Ni-Ti alloy surface (a), Example 1 (b), Example 2 (c), and Example 3 (d), respectively. DETAILED DESCRIPTION

[0035] The present invention is further described below by the following embodiments. It should be understood that the following embodiments are only used to illustrate the present invention, but not to limit the present invention.

[0036] The present invention provides a method for preparing a monomolecular coating containing phosphorylcholine groups with controllable thickness. The preparation method firstly hydroxylates the surface of the substrate by an acid washing method; secondly, a silane coupling agent is grafted; then, the molecular chain extension is controlled by cyclic reaction of butadiene and propylenediamine layer by layer assembly through Michael addition reaction, and the thickness can be increased by 1 to 2 nm per cycle; finally, phosphorylcholine groups are grafted by Schiff base reaction. The core of the present invention is to design the cyclic reaction of butadiene and propylenediamine layer by layer assembly to control the length of the molecular chain, thereby controlling the thickness of the coating.

[0037] The present invention provides a method for preparing a monomolecular coating containing phosphorylcholine groups with controllable thickness, comprising the following steps:

[0038] (1) Surface hydroxylation: Immerse the substrate material in piranha washing solution and stir for 5 to 30 minutes at a temperature of 20 to 60°C. After cleaning, blow dry with nitrogen.

[0039] (2) Surface amination: The substrate is immersed in toluene, and 3-aminopropyltriethoxysilane is added to prepare a solution with a volume fraction of 1% to 10%. The reaction is stirred and refluxed at 20 to 80° C. in a nitrogen atmosphere for 4 to 24 hours. After ultrasonic cleaning with ethanol and deionized water, the substrate surface is vacuum dried to obtain an amino-functionalized substrate surface.

[0040] (3) Grafting double bond functional groups: 1,3-butadiene (or 1,4-pentadiene, isoprene, 1,5-hexadiene) is dissolved in tetrahydrofuran to prepare a solution with a concentration of 1 to 100 mmol / L. The amination-treated substrate is immersed in the tetrahydrofuran solution and stirred in a nitrogen atmosphere for 4 to 24 hours.

[0041] (4) Grafting amino functional groups: Dissolve 1,3-propylenediamine (or ethylenediamine, 1,4-butylenediamine) in tetrahydrofuran to prepare a solution with a concentration of 1 to 100 mmol / L, immerse the substrate grafted with double bond functional groups in the tetrahydrofuran solution, and stir the reaction in a nitrogen atmosphere for 4 to 24 hours.

[0042] (5) Grafting phosphorylcholine groups: dissolve aldehyde phosphorylcholine in anhydrous methanol to prepare a solution with a mass fraction of 1% to 10%. Immerse the amino-functionalized substrate in the methanol solution and stir the reaction in a nitrogen atmosphere for 4 to 24 hours.

[0043] (6) Reductive amination: Sodium borohydride is dissolved in anhydrous methanol to prepare a solution with a concentration of 1 to 100 mmol / L. The substrate grafted with phosphorylcholine groups is immersed in the methanol solution and stirred for reaction for 4 to 24 hours.

[0044] The final coating molecular formula is as follows Figure 1 As shown. Steps (3) and (4) can be repeated for multiple times. With each additional cycle, a molecule (-NH-CH2-CH2-CH2-CH2-NH-CH2-CH2-CH2-) with a length of 1 to 2 nm is added to the molecular chain. Finally, a monomolecular coating containing phosphorylcholine groups with a controllable coating thickness of 1 to 10 nm can be obtained. The coating can be used to modify the surface of in vivo interventional surgical medical devices such as heart stents to improve their biocompatibility and avoid the formation of blood clots.

[0045] The following further examples are given to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific values ​​​​exemplified below.

[0046] Example 1: Preparation of a 1-2 nm thick phosphorylcholine coating

[0047] (1) Hydroxylation: The nickel-titanium alloy substrate material was immersed in piranha washing solution and stirred for 30 minutes at a temperature of 50°C. After washing, it was blown dry with nitrogen.

[0048] (2) Surface amination: The substrate was immersed in toluene, 3-aminopropyltriethoxysilane was added to prepare a 5% solution, stirred and refluxed at 80°C in a nitrogen atmosphere for 6 hours, and then ultrasonically cleaned with ethanol and deionized water, and then vacuum dried to obtain an amino-functionalized substrate surface.

[0049] (3) Grafting phosphorylcholine groups: Aldehyde phosphorylcholine was dissolved in anhydrous methanol to prepare a solution with a mass fraction of 3%. The amino-functionalized substrate was immersed in the methanol solution and stirred in a nitrogen atmosphere for 12 hours.

[0050] (4) Reductive amination: Sodium borohydride was dissolved in anhydrous methanol to prepare a solution with a concentration of 30 mmol / L. The substrate grafted with phosphorylcholine groups was immersed in the methanol solution and stirred for reaction for 12 hours.

[0051] Figure 2 is the molecular formula of the coating obtained in this example.

[0052] Example 2: Preparation of a 4-5 nm thick phosphorylcholine coating

[0053] (1) Hydroxylation: The nickel-titanium alloy substrate material was immersed in piranha washing solution and stirred for 30 minutes at a temperature of 50°C. After washing, it was blown dry with nitrogen.

[0054] (2) Surface amination: The substrate was immersed in toluene, 3-aminopropyltriethoxysilane was added to prepare a 5% solution, stirred and refluxed at 80°C in a nitrogen atmosphere for 6 hours, and then ultrasonically cleaned with ethanol and deionized water, and then vacuum dried to obtain an amino-functionalized substrate surface.

[0055] (3) Grafting double bond functional groups: 1,3-butadiene was dissolved in tetrahydrofuran to prepare a solution with a concentration of 20 mmol / L. The amination-treated substrate was immersed in the tetrahydrofuran solution and stirred in a nitrogen atmosphere for 12 h.

[0056] (4) Grafting amino functional groups: Ethylenediamine was dissolved in tetrahydrofuran to prepare a solution with a concentration of 20 mmol / L. The substrate grafted with double bond functional groups was immersed in the tetrahydrofuran solution and stirred in a nitrogen atmosphere for 12 h.

[0057] (5) Secondary grafting of double bond functional groups: 1,3-butadiene was dissolved in tetrahydrofuran to prepare a solution with a concentration of 20 mmol / L. The substrate was immersed in the tetrahydrofuran solution and stirred in a nitrogen atmosphere for 12 h.

[0058] (6) Secondary grafting of amino functional groups: Ethylenediamine was dissolved in tetrahydrofuran to prepare a solution with a concentration of 20 mmol / L. The grafted substrate was immersed in the tetrahydrofuran solution and stirred in a nitrogen atmosphere for 12 h.

[0059] (7) Grafting phosphorylcholine groups: Aldehyde phosphorylcholine was dissolved in anhydrous methanol to prepare a solution with a mass fraction of 3%. The amino-functionalized substrate was immersed in the methanol solution and stirred in a nitrogen atmosphere for 12 hours.

[0060] (8) Reductive amination: Sodium borohydride was dissolved in anhydrous methanol to prepare a solution with a concentration of 30 mmol / L. The substrate grafted with phosphorylcholine groups was immersed in the methanol solution and stirred for reaction for 12 hours.

[0061] Figure 3 is the molecular formula of the coating obtained in this example.

[0062] Example 3: Preparation of a phosphorylcholine coating with a thickness of 8 to 10 nm

[0063] (1) Hydroxylation: The nickel-titanium alloy substrate material was immersed in piranha washing solution and stirred for 30 minutes at a temperature of 50°C. After washing, it was blown dry with nitrogen.

[0064] (2) Surface amination: The substrate was immersed in toluene, 3-aminopropyltriethoxysilane was added to prepare a 5% solution, stirred and refluxed at 80°C in a nitrogen atmosphere for 6 hours, and then ultrasonically cleaned with ethanol and deionized water, and then vacuum dried to obtain an amino-functionalized substrate surface.

[0065] (3) Grafting double bond functional groups: 1,3-butadiene was dissolved in tetrahydrofuran to prepare a solution with a concentration of 20 mmol / L. The amination-treated substrate was immersed in the tetrahydrofuran solution and stirred in a nitrogen atmosphere for 12 h.

[0066] (4) Grafting amino functional groups: 1,3-propylenediamine was dissolved in tetrahydrofuran to prepare a solution with a concentration of 20 mmol / L. The substrate grafted with double bond functional groups was immersed in the tetrahydrofuran solution and stirred in a nitrogen atmosphere for 12 h.

[0067] (5) Secondary grafting of double bond functional groups: 1,3-butadiene was dissolved in tetrahydrofuran to prepare a solution with a concentration of 20 mmol / L. The substrate was immersed in the tetrahydrofuran solution and stirred in a nitrogen atmosphere for 12 h.

[0068] (6) Secondary grafting of amino functional groups: 1,3-propylenediamine was dissolved in tetrahydrofuran to prepare a solution with a concentration of 20 mmol / L. The grafted substrate was immersed in the tetrahydrofuran solution and stirred in a nitrogen atmosphere for 12 h.

[0069] (7) Tertiary grafting of double bond functional groups: 1,3-butadiene was dissolved in tetrahydrofuran to prepare a solution with a concentration of 20 mmol / L. The substrate was immersed in the tetrahydrofuran solution and stirred for reaction in a nitrogen atmosphere for 12 h.

[0070] (8) Tertiary grafting of amino functional groups: 1,3-propylenediamine was dissolved in tetrahydrofuran to prepare a solution with a concentration of 20 mmol / L. The grafted substrate was immersed in the tetrahydrofuran solution and stirred in a nitrogen atmosphere for 12 h.

[0071] (9) Quadruple grafting of double bond functional groups: 1,3-butadiene was dissolved in tetrahydrofuran to prepare a solution with a concentration of 20 mmol / L. The substrate was immersed in the tetrahydrofuran solution and stirred for reaction in a nitrogen atmosphere for 12 h.

[0072] (10) Quadruple grafting of amino functional groups: 1,3-propylenediamine was dissolved in tetrahydrofuran to prepare a solution with a concentration of 20 mmol / L. The grafted substrate was immersed in the tetrahydrofuran solution and stirred in a nitrogen atmosphere for 12 h.

[0073] (11) Five-time grafting of double bond functional groups: 1,3-butadiene was dissolved in tetrahydrofuran to prepare a solution with a concentration of 20 mmol / L. The substrate was immersed in the tetrahydrofuran solution and stirred in a nitrogen atmosphere for 12 h.

[0074] (12) Five times of grafting amino functional groups: 1,3-propylenediamine was dissolved in tetrahydrofuran to prepare a solution with a concentration of 20 mmol / L. The grafted substrate was immersed in the tetrahydrofuran solution and stirred in a nitrogen atmosphere for 12 h.

[0075] (13) Six-time grafting of double bond functional groups: 1,3-butadiene was dissolved in tetrahydrofuran to prepare a solution with a concentration of 20 mmol / L. The substrate was immersed in the tetrahydrofuran solution and stirred for reaction in a nitrogen atmosphere for 12 h.

[0076] (14) Six-time grafting of amino functional groups: 1,3-propylenediamine was dissolved in tetrahydrofuran to prepare a solution with a concentration of 20 mmol / L. The grafted substrate was immersed in the tetrahydrofuran solution and stirred in a nitrogen atmosphere for 12 h.

[0077] (15) Grafting phosphorylcholine groups: Aldehyde phosphorylcholine was dissolved in anhydrous methanol to prepare a solution with a mass fraction of 3%. The amino-functionalized substrate was immersed in the methanol solution and stirred in a nitrogen atmosphere for 12 h.

[0078] (16) Reductive amination: Sodium borohydride was dissolved in anhydrous methanol to prepare a solution with a concentration of 30 mmol / L. The substrate grafted with phosphorylcholine groups was immersed in the methanol solution and stirred for reaction for 12 hours.

[0079] Figure 4 is the molecular formula of the coating obtained in this example.

[0080] Figure 5 The adhesion of platelets on (a) Ni-Ti alloy surface, (b) coating surface of Example 1, (c) Example 2, and (d) Example 3 after incubation at 37°C for 90 minutes. Figure 5 The green part in (a) is a platelet cell stained with phalloidin fluorescence. Platelet cell adhesion is observed on the blank Ni-Ti alloy surface, and no platelet adhesion is observed on the surface of the coating with different thicknesses containing phosphorylcholine groups at the end, indicating that the phosphorylcholine group coating of the present invention can resist platelet adhesion and has anticoagulant properties.

[0081] Finally, it is necessary to explain here that the above embodiments are only used to further illustrate the technical solution of the present invention in detail and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above content of the present invention all fall within the scope of protection of the present invention.

Claims

1. A method for preparing a thickness-controllable phosphorylcholine monomolecular coating, characterized in that: The preparation method extends the length of the molecule by layer-by-layer assembly through Michael addition reaction, and comprises the following steps: Step (1), hydroxylation of the surface of the substrate material; Step (2), aminate the surface obtained in step (1); in step (2), the surface aminate comprises: immersing the substrate in toluene, adding 3-aminopropyltriethoxysilane to prepare a solution with a volume fraction of 1%-10%; stirring and refluxing at 20-80° C. in a nitrogen atmosphere for 4-24 h, ultrasonically cleaning with ethanol and deionized water, and vacuum drying to obtain an amino-functionalized substrate surface; Step (3), grafting double bond functional groups on the surface obtained in step (2); in step (3), the surface grafting double bond functional groups comprises: dissolving a diene in tetrahydrofuran to prepare a solution with a concentration of 1-100 mmol / L, immersing the amination-treated substrate in the tetrahydrofuran solution, and stirring the reaction in a nitrogen atmosphere for 4-24 hours; the diene is selected from one or more of 1,3-butadiene, 1,4-pentadiene, isoprene, and 1,5-hexadiene; Step (4), grafting amino functional groups on the surface obtained in step (3); in step (4), the grafting of amino functional groups comprises: dissolving a diamino compound in tetrahydrofuran to prepare a solution with a concentration of 1-100 mmol / L, immersing the substrate grafted with double bond functional groups in the tetrahydrofuran solution, and stirring the reaction in a nitrogen atmosphere for 4-24 hours; the diamino compound is selected from one or more of ethylenediamine, 1,3-propylenediamine, and 1,4-butylenediamine; Step (5), grafting phosphorylcholine groups onto the surface obtained in step (4); the surface grafting phosphorylcholine groups comprises: dissolving aldehyde phosphorylcholine in anhydrous methanol to prepare a solution with a mass fraction of 1%-10%; immersing the grafted amino-functional substrate in the methanol solution, and stirring the reaction in a nitrogen atmosphere for 4-24 hours; Step (6), adding a reducing agent to the surface obtained in step (5) to reduce the imine bond; in step (6), reducing the imine bond comprises: dissolving sodium borohydride in anhydrous methanol to prepare a solution with a concentration of 1-100 mmol / L; immersing the substrate grafted with phosphorylcholine groups in the methanol solution, stirring and reacting for 4-24 hours; Wherein, step (3) and step (4) are repeatedly cycled for multiple times to control the coating thickness, thereby obtaining a single-molecule coating with a controllable thickness and containing a phosphorylcholine group at the end; the thickness of the coating is 1-10 nm.

2. The preparation method according to claim 1, characterized in that In step (1), the method for hydroxylating the surface of the substrate material comprises: immersing the substrate material in piranha washing solution and stirring for 5-30 minutes at a temperature of 20-60° C., and drying with nitrogen after washing.

3. A monomolecular phosphorylcholine coating with controllable thickness prepared by the preparation method according to claim 1 or 2.

4. An in vivo interventional surgery medical device, characterized in that: It comprises the thickness-controllable phosphorylcholine monomolecular coating as claimed in claim 3.

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