CN102268639A - Method for preparing heparinized interface material with high biological functionality - Google Patents
Method for preparing heparinized interface material with high biological functionality Download PDFInfo
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- 238000002360 preparation method Methods 0.000 claims abstract description 23
- 238000000151 deposition Methods 0.000 claims abstract description 22
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- Materials For Medical Uses (AREA)
Abstract
The invention discloses a method for preparing a heparinized interface material with high biological functionality. The method comprises the following steps of: depositing a layer of allyl amine plasma polymerization film on the surface of a substrate material subjected to sputter cleaning by adopting a pulse plasma polymerization method, and soaking the biological material deposited with the plasma polymerization film in a phosphate buffer solution (PBS) with the pH value of 2-7 for 1 to 24 hours, so that the amino on the surface is subjected to protonation; and adding heparin sodium into the PBS to ensure that the concentration is 1-10mg/ml, reacting at room temperature of 25DEG C for 1 to 24 hours, fully rinsing by using distilled water, and drying to obtain the target material. In the method, a heparin monomolecular layer is constructed on the surface of the plasma polymerization film by a soaking self-assembly mode only; and the preparation process is easy and convenient to operate, expensive special equipment is not needed and the preparation cost is low.
Description
Technical field
The present invention relates to the biotechnology functional materials, especially have the heparinization boundary material preparing technical field of biological functionality.
Background technology
Heparin is the Sulfated form of the height of Suleparoid, is a kind of biomolecules with anticoagulant property, is present in the extracellular matrix, it also is the specific recognition body of multiple protein enzyme and somatomedin.Therefore, anticoagulating active and specific recognition that interface heparinization material has have obtained extensive studies and application at biochip technology and technical field of biological material.The method for preparing at present the heparinization boundary material mainly contains physical adsorption and Covalent Immobilization.The heparin molecule physical adsorption is in the combination instability of material surface, and situation following very short time has discharged in the body.Heparin is the most popular method of preparation heparinization boundary material in the Covalent Immobilization of material surface, although the heparinization boundary material of Covalent Immobilization has than persistence for the reservation of heparin molecule, yet Covalent Immobilization is weakened the biological activity of heparin molecule greatly, the reduction greatly of its potential protein-specific recognition capability has limited its using value of further expanding.
Summary of the invention
Above deficiency in view of prior art, the purpose of this invention is to provide a kind of method of preparing boundary material with height heparin biological activity confining force, it has in the height biological activity confining force in assurance, it is simple and convenient to make it to have preparation manipulation, need not expensive specific equipment, the advantage that preparation cost is low.
The objective of the invention is to realize by following means.
A kind of heparinization interface material producing method with high biological functionality, adopt following steps to obtain the heparinization boundary material at biomaterial surface:
A, employing pulsed plasma polymerization process are at deposition one deck allylamine plasma polymerization film on the substrate material surface of sputter clean;
It is in the PBS solution of 2-7 1-24 hour that B, the biomaterial that deposits the plasma polymerization film after A handled are soaked in pH value, makes its surface amino groups protonated;
C, heparin sodium is added in the described PBS buffered soln of B step, concentration is 1-10mg/ml, and reaction 1-24 hour down of 25 ℃ of room temperatures after reaction finishes, is used the abundant rinsing of PBS and distilled water respectively, drying, target material.
Adopt the heparinization boundary material of the inventive method preparation not only to have excellent anti-freezing and adhesion of anti-smooth muscle cell and multiplication capacity, and enzyme (as protease A TIII), extracellular matrix protein (as Fn), somatomedin (as VEGF), the chemokine (as SDF-1 α) of its specific recognition shown excellent specific recognition ability.The heparin functionalization boundary material of the proteolytic enzyme (ATIII) of binding specificity identification, extracellular matrix protein albumen (fibronectin), somatomedin (FGF-1, FGF-2, VEGF), chemokine (SDF-1 α) etc. is with a wide range of applications in fields such as anti-freezing, vasculogenesis, immunology, promotion wound healing, tissue regeneration medical science.
Description of drawings
Fig. 1 is the APTT figure as a result of the 316L stainless steel that obtains of the present invention and heparinization boundary material Hep-PPAam.
Fig. 2 is 316L stainless steel and the heparinization boundary material Hep-PPAam quantitative result figure in conjunction with ATIII and VEGF.
Embodiment
Below in conjunction with embodiment enforcement of the present invention is further described, the embodiment agents useful for same is analytical pure.For sake of convenience, " base material " among the application is to adopting the inventive method to carry out the general designation of the material and the device of surface modification biology.
Embodiment 1
The preparation cobalt base alloy is the heparinization boundary material of base material:
A, plasma body polyallylamine depositing of thin film
The A1 sputter clean: base material is put into film deposition chamber, and when the reaction chamber vacuum is evacuated to when being 0.01, feeding flow is the argon gas of 1sccm, is 50W at radio frequency power, and negative bias is the sputter clean of carrying out under 50 the condition 5 minutes;
A2 plasma polymerization film preparation: after the sputter clean, again the vacuum tightness that makes reaction chamber is 0.01Pa, the argon gas that feeds flow and be 0.5sccm is as discharge gas, and feeding is as the allylamine of reactant gases, making operating pressure is 1Pa, radio frequency power 5W, negative bias be 50V, pulse duty factor be carry out under 5% the condition for the time 60 minutes plasma polymerization thin film deposition, promptly deposition is rich in amino plasma polymerization film on substrate material surface.
B, plasma polymerization film surface proton functionalization
It is in 2 the PBS solution 1 hour that the material that A step is deposited the plasma polymerization film is soaked in the pH value, makes its surface amino groups protonated.
C, the self-assembly of heparin unimolecular layer
Heparin sodium is added in the B PBS buffered soln in step, and concentration is 1mg/ml, and room temperature was reacted 1 hour down for 25 ℃, after reaction finishes, used the abundant rinsing of PBS and distilled water respectively, drying, promptly.
Embodiment 2
The preparation iron is the heparinization boundary material of base material:
A, plasma body polyallylamine depositing of thin film
The A1 sputter clean: base material being put into film deposition chamber, when the reaction chamber vacuum is evacuated to 2Pa, feed the argon gas of flow 10sccm, is 200W at radio frequency power, and negative bias is the sputter clean of carrying out under the condition of 150V 30 minutes;
A2 plasma polymerization film preparation: after the sputter clean, again the vacuum tightness that makes reaction chamber is 2Pa, the argon gas that feeds flow and be 5sccm is as discharge gas, and feeding is as the allylamine of reactant gases, make operating pressure 10Pa, radio frequency power 50W, negative bias be 150V, pulse duty factor be carry out under 100% the condition for the time 5 minutes plasma polymerization thin film deposition, promptly deposition is rich in amino plasma polymerization film on substrate material surface.
B, plasma polymerization film surface proton functionalization
It is in 7 the PBS solution 24 hours that the base material that A step is deposited the plasma polymerization film is soaked in the pH value, makes its surface amino groups protonated.
C, the self-assembly of heparin unimolecular layer
Heparin sodium is added in the B PBS buffered soln in step, and concentration is 10mg/ml, and room temperature was descended anti-24 hours for 25 ℃, after reaction finishes, uses the abundant rinsing of PBS and distilled water respectively, drying, promptly.
Embodiment 3
Preparation NiTi alloy is the heparinization boundary material of base material:
A, plasma body polyallylamine depositing of thin film
The A1 sputter clean: base material is put into film deposition chamber, and when the reaction chamber vacuum was evacuated to 0.2Pa, feeding flow was the argon gas of 5sccm, is 100W at radio frequency power, and negative bias is the sputter clean of carrying out under the condition of 100V 15 minutes;
A2 plasma polymerization film preparation: after the sputter clean, again the vacuum tightness that makes reaction chamber is 0.2Pa, the argon gas that feeds flow and be 2sccm is as discharge gas, and feeding allylamine as reactant gases, to make operating pressure be 5Pa, radio frequency power 30W, negative bias be 100V, pulse duty factor be carry out under 30% the condition for the time 30 minutes plasma polymerization thin film deposition, promptly deposition is rich in amino plasma polymerization film on material surface.
B, plasma polymerization film surface proton functionalization
It is in 4 the PBS solution 12 hours that the material that A step is deposited the plasma polymerization film is soaked in the pH value, makes its surface amino groups protonated.
C, the self-assembly of heparin unimolecular layer
Heparin sodium is added in the B PBS buffered soln in step, and concentration is 5mg/ml, and room temperature was reacted 12 hours down for 25 ℃, after reaction finishes, used the abundant rinsing of PBS and distilled water respectively, drying, promptly.
Embodiment 4
The preparation titanium alloy is the heparinization boundary material of base material:
A, plasma body polyallylamine depositing of thin film
The A1 sputter clean: material is put into film deposition chamber, and when the reaction chamber vacuum was evacuated to 1Pa, feeding flow was the argon gas of 8sccm, is 150W at radio frequency power, and negative bias is the sputter clean of carrying out under the condition of 75V 25 minutes;
A2 plasma polymerization film preparation: after the sputter clean, again the vacuum tightness that makes reaction chamber is 1Pa, the argon gas that feeds flow and be 4sccm is as discharge gas, and feeding is as the allylamine of reactant gases, making operating pressure is 8Pa, radio frequency power 40W, negative bias be 120V, pulse duty factor be carry out under 50% the condition for the time 45 minutes plasma polymerization thin film deposition, promptly deposition is rich in amino plasma polymerization film on the intravascular stent surface.
B, plasma polymerization film surface proton functionalization
It is in 5.5 the PBS solution 1-24 hour that the material that A step is deposited the plasma polymerization film is soaked in the pH value, makes its surface amino groups protonated.
C, the self-assembly of heparin unimolecular layer
Heparin sodium is added in the B PBS buffered soln in step, and concentration is 8mg/ml, and room temperature was reacted 20 hours down for 25 ℃, after reaction finishes, used the abundant rinsing of PBS and distilled water respectively, drying, promptly.
D, ATIII fix
The material that C is gone on foot surface heparinization is immersed in the PBS solution that contains antiprothrombin (ATIII), and room temperature was reacted 24 hours down for 25 ℃, after reaction finishes, used the abundant rinsing of PBS and distilled water respectively, drying, promptly.The APTT result of its Hep-PPAam such as Fig. 1.
Embodiment 5
Preparation 316L stainless steel is the heparinization boundary material of base material:
A, plasma body polyallylamine depositing of thin film
The A1 sputter clean: base material is put into film deposition chamber, and when the reaction chamber vacuum was evacuated to 0.01Pa, feeding flow was the argon gas of 10sccm, is 200W at radio frequency power, and negative bias is the sputter clean of carrying out under the condition of 150V 30 minutes;
A2 plasma polymerization film preparation: after the sputter clean, again the vacuum tightness that makes reaction chamber is 0.01Pa, the argon gas that feeds flow and be 5sccm is as discharge gas, and feeding is as the allylamine of reactant gases, making operating pressure is 10Pa, radio frequency power 50W, negative bias be 150V, pulse duty factor be carry out under 5% the condition for the time 60 minutes plasma polymerization thin film deposition, promptly deposition is rich in amino plasma polymerization film on material surface.
B, plasma polymerization film surface proton functionalization
It is in 2 the PBS solution 24 hours that the material that A step is deposited the plasma polymerization film is soaked in the pH value, makes its surface amino groups protonated.
C, the self-assembly of heparin unimolecular layer
Heparin sodium is added in the B PBS buffered soln in step, and concentration is 10mg/ml, and room temperature was reacted 24 hours down for 25 ℃, after reaction finishes, used the abundant rinsing of PBS and distilled water respectively, drying, promptly.
D, VEGF's is fixing
The material that C is gone on foot surface heparinization is immersed in the PBS solution that contains endothelial cell growth factor (ECGF) (VEGF), and room temperature was reacted 24 hours down for 25 ℃, after reaction finishes, used the abundant rinsing of PBS and distilled water respectively, and drying promptly gets target product.Its Hep-PPAam in conjunction with the quantitative result of ATIII and VEGF as shown in Figure 2.
Fig. 1 has improved the endogenous anticoagulation function significantly for the partial thromboplastin time result with the heparinization boundary material of method of the present invention preparation shows heparinization boundary material surface, and the anticoagulant property that heparinization boundary material that this patent prepares has excellence be described.
Fig. 2 for the heparinization boundary material for preparing with method of the present invention to AT-III and VEGF specificity bonded quantitative test result, show that this change boundary material can significantly promote the binding capacity to AT-III and VEGF, illustrate that the heparinization boundary material of this patent preparation has excellent excellent heparin-protein binding function.
The good result of Fig. 1 and Fig. 2 also is without loss of generality to other embodiment of the present invention.
Preparation method of the present invention makes up the heparin unimolecular layer with height biological activity confining force at biomaterial and apparatus surface thereof.The using plasma polymerization technique at biomaterial and apparatus surface deposition last layer thereof in conjunction with firmly and be rich in the plasma polymerization film of amidine functional group.After soaking in acid PBS, surface amino groups is by protonated and with going up abundant positive charge, strong electrostatic interaction takes place with heparin molecule self negative electricity group (carboxylate radical and sulfonate radical) after, effectively heparin in conjunction with last its surface.
Compared with prior art, the invention has the beneficial effects as follows:
One, polymer coated and biomaterial and apparatus surface employing plasma deposition method thereof, in conjunction with firm, coating contains abundant amido functional group.
Two, rely on the strong electrostatic adsorption of the protonated amino of the carboxylate radical of heparin and sulfonate radical and plasma polymerization film surface to have very strong bonding force at its surperficial fixed heparin.
Three, do not adopt any coupling agent because heparin is fixing, make that heparin is functional effectively to be kept, show excellent heparin-protein binding function.In albumen, biomolecules and the somatomedin of this heparin functionalization boundary material surface bonding specific recognition, can realize its application by further in fields such as anti-freezing, vasculogenesis, immunology, promotion wound healing, tissue regeneration medical science.
The preparation of this kind heparin functionalization boundary material simultaneously, only need with soak self-assembly mode make up the heparin unimolecular layer at the plasma polymerization film surface, its preparation process, simple to operation, also need not expensive specific equipment, preparation cost is low.
Adopt basic skills of the present invention, the actual widely example that becomes also can be arranged when reality is implemented, and for example " base material " among the application can comprise bio-medical metal_based material (Fe, mg-based material, 316L SS, Ti, Ti alloy Ni-Ti alloy and Co-Cr alloy etc.), inorganic materials (Ti-O, TiN etc.), non-degradable polymkeric substance (as: PET, PTFE, PDMS etc.) and degradable polymer material (as PLA, PLGA and PCL etc.).
Claims (4)
1. heparinization interface material producing method with high biological functionality, adopt following steps to obtain the heparinization boundary material at biomaterial surface:
A, employing pulsed plasma polymerization process are at deposition one deck allylamine plasma polymerization film on the substrate material surface of sputter clean;
It is in the PBS solution of 2-7 1-24 hour that B, the biomaterial that deposits the plasma polymerization film after A handled are soaked in pH value, makes its surface amino groups protonated;
C, heparin sodium is added in the described PBS buffered soln of B step, concentration is 1-10mg/ml, and reaction 1-24 hour down of 25 ℃ of room temperatures after reaction finishes, is used the abundant rinsing of PBS and distilled water respectively, drying, target material.
2. a kind of heparinization interface material producing method according to claim 1 with high biological functionality, it is characterized in that the described A step with pulsed plasma polymerization concrete practice of deposition plasma polymer thin film on bio-medical material and device surface thereof is:
A1 sputter clean: bio-medical material and device surface thereof are put into film deposition chamber, when the reaction chamber vacuum is evacuated to 0.01-2Pa, feeding flow is the argon gas of 1-10sccm, at radio frequency power is 50-200W, and negative bias is the sputter clean of carrying out under the condition of 50-150V 5-30 minute;
A2 plasma polymerization film preparation: after the sputter clean, again the vacuum tightness that makes reaction chamber is 0.01-2Pa, the argon gas that feeds flow and be 0.5-5sccm is as discharge gas, and feeding is as the allylamine of reactant gases, making operating pressure is 1-10Pa, radio frequency power 5-50W, negative bias be 50-150V, pulse duty factor be carry out under the condition of 5-100% for the time 5-60 minute plasma polymerization thin film deposition, obtain described allylamine plasma polymerization film.
3. a kind of heparinization interface material producing method with high biological functionality according to claim 1 is characterized in that described base material can be the bio-medical metal_based material.
4. a kind of heparinization interface material producing method according to claim 3 with high biological functionality, it is characterized in that described bio-medical metal_based material is 316L stainless steel, titanium and alloy thereof, NiTi alloy, cobalt base alloy, iron and mg-based material.
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| CN103614718A (en) * | 2013-10-30 | 2014-03-05 | 西南交通大学 | Method for obtaining bio-functionalized coating with low corrosion speed on pure magnesium surface |
| CN107224621A (en) * | 2016-03-23 | 2017-10-03 | 成都交大麦迪克科技有限公司 | One kind has endothelium bionic function coating and preparation method thereof |
| CN107812253A (en) * | 2017-11-17 | 2018-03-20 | 南通纺织丝绸产业技术研究院 | A kind of test tube of hepari fibroin protein film and preparation method thereof |
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| CN101361992A (en) * | 2008-09-12 | 2009-02-11 | 西南交通大学 | Method for fixing heparin multilayer film on the surface of titanium dioxide |
| CN101927037A (en) * | 2009-12-10 | 2010-12-29 | 西南交通大学 | Method for preparing anticoagulant vascular stent |
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| CN101361992A (en) * | 2008-09-12 | 2009-02-11 | 西南交通大学 | Method for fixing heparin multilayer film on the surface of titanium dioxide |
| CN101927037A (en) * | 2009-12-10 | 2010-12-29 | 西南交通大学 | Method for preparing anticoagulant vascular stent |
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| 《功能材料》 20081231 杨志禄等 等离子体聚合沉积聚烯丙胺薄膜改性聚酯及体外血小板粘附研究 1559-1566页 1-4 第39卷, 第9期 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103614718A (en) * | 2013-10-30 | 2014-03-05 | 西南交通大学 | Method for obtaining bio-functionalized coating with low corrosion speed on pure magnesium surface |
| CN103614718B (en) * | 2013-10-30 | 2016-02-03 | 西南交通大学 | A kind of method obtaining the coating of biological functional and low corrosion speed at pure magnesium surface |
| CN107224621A (en) * | 2016-03-23 | 2017-10-03 | 成都交大麦迪克科技有限公司 | One kind has endothelium bionic function coating and preparation method thereof |
| CN107812253A (en) * | 2017-11-17 | 2018-03-20 | 南通纺织丝绸产业技术研究院 | A kind of test tube of hepari fibroin protein film and preparation method thereof |
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