CN103721300A - Anticoagulation coating material and preparation method thereof - Google Patents
Anticoagulation coating material and preparation method thereof Download PDFInfo
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- CN103721300A CN103721300A CN201310700680.3A CN201310700680A CN103721300A CN 103721300 A CN103721300 A CN 103721300A CN 201310700680 A CN201310700680 A CN 201310700680A CN 103721300 A CN103721300 A CN 103721300A
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Abstract
The invention discloses an anticoagulation coating material and a preparation method thereof. The anticoagulation coating material comprises sodium hyaluronate and hirudin, wherein the sodium hyaluronate is formed into a gel network; the hirudin is in covalent binding with the sodium hyaluronate. The preparation method of the anticoagulation coating material comprises the following steps: (1) preparing an MES (Fatty Acid Methyl Ester Sulfonate) buffer solution; (2) preparing a sodium hyaluronate solution with the concentration of 1mg/ml to 20mg/ml; (3) preparing a hirudin solution with the concentration of 1mg/ml to 10mg/ml; (4) uniformly mixing up the two solutions so as to enable the mass ratio of the hirudin to the sodium hyaluronate to be 1:1 to 1:10; (5) adding a crosslinking agent into the mixture solution obtained in the step (4), uniformly mixing up so as to covalently crosslink the hirudin and the sodium hyaluronate. The anticoagulation coating material disclosed by the invention is high in anticoagulation activity, stable in effect, long in lasting time, good in biocompatibility, simple in preparation method, gentle in condition and applicable to large-scale industrial production.
Description
Technical field
The invention belongs to biomedical materials field, more specifically, relate to a kind of anticoagulant coatings material and preparation method thereof.
Background technology
Anticoagulation bio-medical material is the important biomaterial of a class, on the medical material that is widely used in contacting with human blood and tissue, as hemodialysis system, extracorporeal circulation system, Cardiac valve prosthesis, cardiac pacemaker, artificial blood vessel, intravascular stent, surgical cable and conduit etc.Enter 21 century, cardiovascular disease incidence rate and mortality rate are in continuous rising, and the control of cardiovascular disease has become the emphasis that domestic and international medical circle is paid close attention to.Anticoagulation bio-medical material has important using value.
At present existing anticoagulant coatings, is mainly to adopt heparin as anticoagulin, and heparin is fixed on to material surface by physical absorption or chemically combined method.The method that adopts physical absorption, by material heparinization, heparin easily runs off, and anticoagulant effect is not long; Adopt chemically combined method, by material heparinization, because heparin is mcroorganism molecule, covalently bind in after metal support surface, its conformation is restricted, and may cause anticoagulant active to reduce.For the coated polymeric of fixing heparin, also there is biocompatibility issues in addition, can not promote endothelium healing, to reducing also not obviously contribution of restenosis rate.
Also there is no at present a kind of anticoagulant coatings material that there is desirable blood coagulation resisting function and do not affect its biocompatibility.
Summary of the invention
For above defect or the Improvement requirement of prior art, the invention provides a kind of anticoagulant coatings material, its object is by by anticoagulin hirudin stable to molecular weight, anticoagulant effect and the hyaluronic acid sodium gel network covalent bond with good biocompatibility, solves thus current anticoagulant coatings material anticoagulant effect unstable or permanent, the poor technical problem of biocompatibility.
For achieving the above object, according to one aspect of the present invention, provide a kind of anticoagulant coatings material, comprised hyaluronate sodium and hirudin, described hyaluronate sodium forms gel network, and described hirudin is by amido link and described hyaluronate sodium covalent bond.
Preferably, described anticoagulant coatings material, the mass ratio of its hirudin and described hyaluronate sodium is 1:1 to 1:10.
Preferably, described anticoagulant coatings material, the mass ratio of its hirudin and described hyaluronate sodium is 1:3 to 1:5.
Preferably, described anticoagulant coatings material, also comprises cross-linking agent.
Preferably, described anticoagulant coatings material, its cross-linking agent is 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides and N-hydroxy-succinamide.
According to another aspect of the present invention, a kind of preparation method of anticoagulant coatings material is provided, comprise the following steps:
(1) preparation 2-(N-morpholine) ethyl sulfonic acid (MES) buffer solution (0.1mol/L, pH=6.4).
(2) the MES buffer solution that uses step (1) to prepare, adds hyaluronate sodium, and concentration is 1mg/ml to 20mg/ml, fully dissolves and makes sodium hyaluronate solution;
(3) the MES buffer solution that uses step (1) to prepare, adds hirudin, and concentration is 1mg/ml to 10mg/ml, fully dissolves and makes hirudin solution;
(4) the hirudin solution of preparation in the sodium hyaluronate solution of preparation in step (2) and step (3) is evenly mixed, the mass ratio that makes hirudin and hyaluronate sodium is 1:1 to 1:10;
(5) in the mixed solution obtaining in step (4), add cross-linking agent, evenly mix, make hirudin and hyaluronate sodium covalent cross-linking, make anticoagulant coatings material.
Preferably, described anticoagulant coatings material preparation method, it is 0.1% to 1.0% Tween 80 that the hirudin solution in its step (3) contains mass concentration.
Preferably, described anticoagulant coatings material preparation method, the cross-linking agent using in its step (5) is freshly prepared 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides solution and N-hydroxy-succinamide solution.
Preferably, described anticoagulant coatings material preparation method, the mass ratio of its hirudin, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides and N-hydroxy-succinamide is 1:5:1.
Preferably, described anticoagulant coatings material preparation method, also comprises:
(6) anticoagulant coatings material step (5) being made is dialysed, and removes cross-linking agent.
In general, the above technical scheme of conceiving by the present invention compared with prior art, owing to adopting molecular weight hirudin and the good hyaluronate sodium cross-linking agent of biocompatibility as anticoagulant coatings material, can obtain following beneficial effect:
(1) hirudin that includes the direct Trombin inhibiting of energy in described anticoagulant coatings material is as anticoagulant material.Hirudin causes thrombin to lose activity by the Binding Capacity group that blocks thrombin, the combination by electrostatic attraction of its free hydroxyl and thrombin free amino group, the complex producing can not dissociate within thrombin keeps active whole pH, and therefore hirudin has that anticoagulant active is high, Stability Analysis of Structures, is difficult for the feature of inactivation.Therefore described anticoagulant coatings material anticoagulant active is higher, is difficult for because conformation change causes anticoagulin inactivation, and anticoagulant effect is stable.Hyaluronate sodium, separately as coating, has certain blood coagulation resisting function, and as shown in Figure 2, platelet adhesion reaction quantity is at 1200/mm
2to 2000/mm
2between, lower than normal level, the hyaluronate sodium coating after hirudin is modified provided by the invention, can bring into play significant anticoagulant effect, and platelet adhesion reaction quantity drops to 500/mm
2below, as shown in Figure 1.Anticoagulant coatings material provided by the invention can be brought into play significant anticoagulant effect as can be seen here.
(2), because hirudin is small-molecular peptides, therefore separately as anticoagulant coatings material, run off and seriously cause anticoagulation time short.The present invention adopts stronger crosslinked of viscosity to be the netted fixing hirudin of hyaluronate sodium, and hirudin and hyaluronate sodium pass through amido link covalent bond, form stable gel network, sustainable permanent performance anticoagulant efficacy.
(3) hyaluronate sodium can provide high-hydrophilic top layer, be applicable to the surface contacting with blood for a long time, there is certain anticoagulant effect and good biocompatibility simultaneously, can with hirudin synergism, strengthen its anticoagulant effect, use anticoagulant coatings material provided by the invention, implant is transformed, make implant itself there is anticoagulant effect steady in a long-term, can help to reduce the use of anticoagulant, thereby avoid as far as possible or alleviate the toxic and side effects of anticoagulant.
(4) anticoagulant coatings material preparation method provided by the invention, for hirudin glutamic acid and the higher feature of aspartic acid content, adopt amido link to make hirudin and hyaluronate sodium covalent bond, can make hirudin be combined with hyaluronate sodium stable, thereby make anticoagulant coatings material bring into play permanent anticoagulant effect.There is no at present the method that bibliographical information can make to form between hirudin and hyaluronate sodium stable bond.
(5) due to anticoagulant coatings material preparation method provided by the invention, adopt cross-linking agent to help to form amido link, can significantly improve reaction efficiency, and selected cross-linking agent can remove by dialysis, residual few, almost there is no toxic and side effects.
(6) anticoagulant coatings material preparation method provided by the invention, technique is comparatively simple, and reaction condition gentleness is applicable to producing in enormous quantities.
Preferred version, kind by the mass ratio range to hirudin and hyaluronate sodium, cross-linking agent and cross-linking agent adding proportion are done and are optimized, anticoagulant coatings material is averaged out between anticoagulant effect, preparation difficulty, production time and production cost, obtain cost comparatively cheap, produce relatively simply, the anticoagulant coatings material that anticoagulant effect is good, anticoagulation time is long.
Accompanying drawing explanation
Fig. 1 is the electron scanning micrograph that anticoagulant coatings material provided by the invention carries out platelet adhesion reaction test;
Fig. 2 is that hyaluronate sodium coating is carried out the electron scanning micrograph after platelet adhesion test.
The specific embodiment
In order to make object of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.In addition,, in each embodiment of described the present invention, involved technical characterictic just can combine mutually as long as do not form each other conflict.
Hyaluronate sodium is the hyaluronic sodium salt of a kind of acid mucopolysaccharide.Hyaluronic acid is widely distributed in the various soft tissues of human body, can form gel, hyaluronic acid has height viscoelasticity, permeability and the rheological properties of uniqueness and good biocompatibility, and gel or a kind of absorbable biological material of hyaluronic acid formation.
Hirudin is a kind of low molecular weight polypeptide being comprised of 65 aminoacid, and the content of its Glutamic Acid and asparagine is higher, and molecular weight is 7000 left and right, contains three disulfide bond.Hirudin is combined with the free amino group of thrombin by its free hydroxyl, cause thrombin to lose activity, within the scope of active pH in this combination at thrombin, can not dissociate, therefore hirudin has that anticoagulant active is high, Stability Analysis of Structures, is difficult for the features such as inactivation, in addition, hirudin later stage purification cost is low, and these advantages make hirudin become the desirable feedstock of a kind of anticoagulant, thrombolytic.
A kind of anticoagulant coatings material provided by the invention, comprises hyaluronate sodium and hirudin, and described hyaluronate sodium forms gel network, and described hirudin is by amido link and described hyaluronate sodium covalent bond.The mass ratio of described hirudin and described hyaluronate sodium is 1:1 to 1:10, and preferable range is 1:3 to 1:5.In order to shorten cross-linking reaction time, promote amido link to form, described anticoagulant coatings material also comprises cross-linking agent, as 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC) and N-hydroxy-succinamide (NHS).
The preparation method of a kind of anticoagulant coatings material provided by the invention, comprises the following steps:
(1) preparation 2-(N-morpholine) ethyl sulfonic acid (MES) buffer solution, concentration is 0.1mol/L, regulates pH value to 6.4.
(2) preparation sodium hyaluronate solution:
Get hyaluronate sodium (HA, molecular weight approximately 2,000,000) and, in 2-(N-morpholine) ethyl sulfonic acid buffer solution (MES, 0.1M, pH=6.4), it is fully dissolved and obtain sodium hyaluronate solution, sodium hyaluronate solution concentration is 1mg/ml to 20mg/ml.Can adopt magnetic agitation to accelerate hyaluronate sodium and dissolve, stir 12 hours.Salable freezing the saving backup of sodium hyaluronate solution making.
(3) preparation hirudin solution:
Get hirudin in MES buffer, hirudin is dispersed in MES buffer.For increasing the dissolubility of hirudin, can add surfactant, for example mass ratio is 0.1% to 1.0% Tween 80.Salable freezing the saving backup of hirudin solution making.
(4) the hirudin solution of preparation in the sodium hyaluronate solution of preparation in step (2) and step (3) is evenly mixed:
In MES buffer, add the sodium hyaluronate solution making in step (2), then add hirudin solution, the mass ratio that makes hirudin and hyaluronate sodium is 1:1 to 1:10, mix homogeneously.
(5) to adding cross-linking agent in the mixed solution obtaining in step (4):
Select 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC) solution and N-hydroxy-succinamide (NHS) solution as cross-linking agent, i.e. EDC/NHS cross-linking agent.Get the EDC solution of freshly prepared 50mg/ml, add in the mixed solution obtaining in step (4), additional proportion is hirudin and EDC mass ratio 1:5, after mix homogeneously standing 15 minutes.Then the NHS solution of getting freshly prepared 10mg/ml, adds described mixed solution, and additional proportion is hirudin and NHS mass ratio 1:1.Under lucifuge condition, evenly mix, 12 as a child obtained hyaluronate sodium and hirudin crosslinking mixture, made described anticoagulant coatings material.
(6) anticoagulant coatings material step (5) being made is dialysed, and removes cross-linking agent, thus purification anticoagulant coatings material.
Anticoagulant coatings material provided by the invention, modifies and normal temperature drying with dip coating, can modify the coating with anticoagulation function.Anticoagulant coatings material provided by the invention is carried out to platelet adhesion reaction test, and after test, with the imaging of surface sweeping ultramicroscope, photo as shown in Figure 1.Only adopt hyaluronate sodium coating material to carry out platelet adhesion reaction test, its test result as shown in Figure 2.Test result is contrasted, can be observed, anticoagulant coatings material provided by the invention has been brought into play significant anticoagulant effect.
Be below embodiment:
Embodiment 1
A kind of anticoagulant coatings material, comprises hyaluronate sodium and hirudin, and described hyaluronate sodium forms gel network, and described hirudin is by amido link and described hyaluronate sodium covalent bond.The mass ratio of described hirudin and described hyaluronate sodium is 1:1.Described anticoagulant coatings material also comprises cross-linking agent, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC) and N-hydroxy-succinamide (NHS).
The preparation method of described anticoagulant coatings material, comprises the following steps:
(1) preparation 2-(N-morpholine) ethyl sulfonic acid (MES) buffer solution, concentration is 0.1mol/L, regulates pH value to 6.4.
(2) preparation sodium hyaluronate solution.
Get hyaluronate sodium (HA, molecular weight approximately 2,000,000) and, in 2-(N-morpholine) ethyl sulfonic acid buffer solution (MES, 0.1M, pH=6.4), it is fully dissolved and obtain sodium hyaluronate solution, sodium hyaluronate solution concentration is 1mg/ml.Can adopt magnetic agitation to accelerate hyaluronate sodium and dissolve, stir 12 hours.Salable freezing the saving backup of sodium hyaluronate solution making.
(3) preparation hirudin solution.
Get hirudin in MES buffer, hirudin concentration is 1mg/ml, by magnetic agitation, hirudin is dispersed in MES buffer, and adding mass concentration is 0.1% Tween 80.Salable freezing the saving backup of hirudin solution making.
(4) the hirudin solution of preparation in the sodium hyaluronate solution of preparation in step (2) and step (3) is evenly mixed.
In 6mL MES buffer, add the sodium hyaluronate solution 20mL making in step (2), the hirudin solution 20mL that then adds step (3) to make, the mass ratio of hyaluronate sodium and hirudin is 1:1, is uniformly mixed.
(5) in the mixed solution obtaining in step (4), add cross-linking agent.
Select 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC) solution and N-hydroxy-succinamide (NHS) solution as cross-linking agent, i.e. EDC/NHS cross-linking agent.Get the EDC solution 2mL of freshly prepared 50mg/ml, add in the mixed solution obtaining in step (4), hirudin and EDC mass ratio 1:5, after mix homogeneously standing 15 minutes.Then the NHS solution 2mL that gets freshly prepared 10mg/ml, adds described mixed solution, hirudin and NHS mass ratio 1:1.Under lucifuge condition, evenly mix, 12 as a child obtained hyaluronate sodium and hirudin crosslinking mixture, made described anticoagulant coatings material.
(6) anticoagulant coatings material step (5) being made is dialysed, and removes cross-linking agent, thus purification anticoagulant coatings material.
Described anticoagulant coatings material employing is soaked to Tu method and be coated on test material polyethylene (PE) sheet, polythene strip is done to infrared spectrum and detect, observe 1650-1750cm
-1and 1200-1320cm (C=O)
-1(C-N) amide (R1-CO-NH-R2) peak strengthens, and illustrates that hyaluronate sodium and hirudin generation amido link are crosslinked.
Described anticoagulant coatings material is carried out to anticoagulation test:
By described anticoagulant coatings material, adopt dip coating to be coated on polythene strip, polythene strip is cut into the square of 2cm*2cm, by centrifugal fresh blood collection platelet rich plasma, be inoculated on the polythene strip that has applied anticoagulant coatings, hatch after fixing dehydration, by sem observation platelet adhesion situation, by the hyaluronate sodium coating of unmodified hirudin as a control group, result shows, the unit are platelet adhesion quantity of the hyaluronate sodium coating of modifying hirudin be do not add the hyaluronate sodium coating of hirudin sample 45%, after covalent bond hirudin, improved the anticoagulation ability of coating.
Embodiment 2
A kind of anticoagulant coatings material, comprises hyaluronate sodium and hirudin, and described hyaluronate sodium forms gel network, and described hirudin is by amido link and described hyaluronate sodium covalent bond.The mass ratio of described hirudin and described hyaluronate sodium is 1:3.Described anticoagulant coatings material also comprises cross-linking agent, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC) and N-hydroxy-succinamide (NHS).
The preparation method of described anticoagulant coatings material, comprises the following steps:
(1) preparation 2-(N-morpholine) ethyl sulfonic acid (MES) buffer solution, concentration is 0.1mol/L, regulates pH value to 6.4.
(2) preparation sodium hyaluronate solution.
Get hyaluronate sodium (HA, molecular weight approximately 2,000,000) and, in 2-(N-morpholine) ethyl sulfonic acid buffer solution (MES, 0.1M, pH=6.4), it is fully dissolved and obtain sodium hyaluronate solution, sodium hyaluronate solution concentration is 3mg/ml.Can adopt magnetic agitation to accelerate hyaluronate sodium and dissolve, stir 12 hours.Salable freezing the saving backup of sodium hyaluronate solution making.
(3) preparation hirudin solution.
Get hirudin in MES buffer solution, hirudin concentration is 1mg/ml, by magnetic agitation, hirudin is dispersed in MES buffer, and adding mass concentration is 0.1% Tween 80.Salable freezing the saving backup of hirudin solution making.
(4) the hirudin solution of preparation in the sodium hyaluronate solution of preparation in step (2) and step (3) is evenly mixed.
In 6mL MES buffer, add the sodium hyaluronate solution 20mL making in step (2), the hirudin solution 20mL that then adds step (3) to make, the mass ratio of hirudin and hyaluronate sodium is 1:3, is uniformly mixed.
(5) in the mixed solution obtaining in step (4), add cross-linking agent.
Select 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC) solution and N-hydroxy-succinamide (NHS) solution as cross-linking agent, i.e. EDC/NHS cross-linking agent.Get the EDC solution 2mL of freshly prepared 50mg/ml, add in the mixed solution obtaining in step (4), hirudin and EDC mass ratio 1:5, after mix homogeneously standing 15 minutes.Then the NHS solution 2mL that gets freshly prepared 10mg/ml, adds described mixed solution, hirudin and NHS mass ratio 1:1.Under lucifuge condition, evenly mix, 12 as a child obtained hyaluronate sodium and hirudin crosslinking mixture, made described anticoagulant coatings material.
Described anticoagulant coatings material employing is soaked to Tu method and be coated on test material polyethylene (PE) sheet, polythene strip is done to infrared spectrum and detect, observe 1650-1750cm
-1and 1200-1320cm (C=O)
-1(C-N) amide (R1-CO-NH-R2) peak strengthens, and illustrates that hyaluronate sodium and hirudin generation amido link are crosslinked.
Described anticoagulant coatings material is carried out to anticoagulation test:
By described anticoagulant coatings material, adopt dip coating to be coated on polythene strip, polythene strip is cut into the square of 2cm*2cm, by centrifugal fresh blood collection platelet rich plasma, be inoculated on the polythene strip that has applied anticoagulant coatings, hatch after fixing dehydration, by sem observation platelet adhesion situation, by the hyaluronate sodium coating of unmodified hirudin as a control group, result shows, the unit are platelet adhesion quantity of the hyaluronate sodium coating of modifying hirudin be do not add the hyaluronate sodium coating of hirudin sample 23%, after covalent bond hirudin, improved the anticoagulation ability of coating.
Embodiment 3
A kind of anticoagulant coatings material, comprises hyaluronate sodium and hirudin, and described hyaluronate sodium forms gel network, and described hirudin is by amido link and described hyaluronate sodium covalent bond.The mass ratio of described hirudin and described hyaluronate sodium is 1:4.Described anticoagulant coatings material also comprises cross-linking agent, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC) and N-hydroxy-succinamide (NHS).
The preparation method of described anticoagulant coatings material, comprises the following steps:
(1) preparation 2-(N-morpholine) ethyl sulfonic acid (MES) buffer solution, concentration is 0.1mol/L, regulates pH value to 6.4.
(2) preparation sodium hyaluronate solution.
Get hyaluronate sodium (HA, molecular weight approximately 2,000,000) and, in 2-(N-morpholine) ethyl sulfonic acid buffer solution (MES, 0.1M, pH=6.4), it is fully dissolved and obtain sodium hyaluronate solution, sodium hyaluronate solution concentration is 5mg/ml.Can adopt magnetic agitation to accelerate hyaluronate sodium and dissolve, stir 12 hours.Salable freezing the saving backup of sodium hyaluronate solution making.
(3) preparation hirudin solution.
Get hirudin in MES buffer solution, hirudin concentration is 2.5mg/ml, by magnetic agitation, hirudin is dispersed in MES buffer, and adding mass concentration is 0.5% Tween 80.Salable freezing the saving backup of hirudin solution making.
(4) the hirudin solution of preparation in the sodium hyaluronate solution of preparation in step (2) and step (3) is evenly mixed.
In 6mL MES buffer, add the sodium hyaluronate solution 8mL making in step (2), the hirudin solution 4mL that then adds step (3) to make, the mass ratio of hirudin and hyaluronate sodium is 1:4, is uniformly mixed.
(5) in the mixed solution obtaining in step (4), add cross-linking agent.
Select 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC) solution and N-hydroxy-succinamide (NHS) solution as cross-linking agent, i.e. EDC/NHS cross-linking agent.Get the EDC solution 1mL of freshly prepared 50mg/ml, add in the mixed solution obtaining in step (4), hirudin and EDC mass ratio 1:5, after mix homogeneously standing 15 minutes.Then the NHS solution 1mL that gets freshly prepared 10mg/ml, adds described mixed solution, hirudin and NHS mass ratio 1:1.Under lucifuge condition, evenly mix, 12 as a child obtained hyaluronate sodium and hirudin crosslinking mixture, made described anticoagulant coatings material.
(6) anticoagulant coatings material step (5) being made is dialysed, and removes cross-linking agent, thus purification anticoagulant coatings material.
Described anticoagulant coatings material employing is soaked to Tu method and be coated on test material polyethylene (PE) sheet, polythene strip is done to infrared spectrum and detect, observe 1650-1750cm
-1and 1200-1320cm (C=O)
-1(C-N) amide (R1-CO-NH-R2) peak strengthens, and illustrates that hyaluronate sodium and hirudin generation amido link are crosslinked.
Described anticoagulant coatings material is carried out to anticoagulation test:
By described anticoagulant coatings material, adopt dip coating to be coated on polythene strip, polythene strip is cut into the square of 2cm*2cm, by centrifugal fresh blood collection platelet rich plasma, be inoculated on the polythene strip that has applied anticoagulant coatings, hatch after fixing dehydration, by sem observation platelet adhesion situation, by the hyaluronate sodium coating of unmodified hirudin as a control group, result shows, the unit are platelet adhesion quantity of the hyaluronate sodium coating of modifying hirudin be do not add the hyaluronate sodium coating of hirudin sample 25%, after covalent bond hirudin, improved the anticoagulation ability of coating.
Embodiment 4
A kind of anticoagulant coatings material, comprises hyaluronate sodium and hirudin, and described hyaluronate sodium forms gel network, and described hirudin is by amido link and described hyaluronate sodium covalent bond.The mass ratio of described hirudin and described hyaluronate sodium is 1:5.Described anticoagulant coatings material also comprises cross-linking agent, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC) and N-hydroxy-succinamide (NHS).
The preparation method of described anticoagulant coatings material, comprises the following steps:
(1) preparation 2-(N-morpholine) ethyl sulfonic acid (MES) buffer solution, concentration is 0.1mol/L, regulates pH value to 6.4.
(2) preparation sodium hyaluronate solution:
Get hyaluronate sodium (HA, molecular weight approximately 2,000,000) and, in 2-(N-morpholine) ethyl sulfonic acid buffer solution (MES, 0.1M, pH=6.4), it is fully dissolved and obtain sodium hyaluronate solution, sodium hyaluronate solution concentration is 10mg/ml.Can adopt magnetic agitation to accelerate hyaluronate sodium and dissolve, stir 12 hours.Salable freezing the saving backup of sodium hyaluronate solution making.
(3) preparation hirudin solution.
Get hirudin in MES buffer solution, hirudin concentration is 5mg/ml, by magnetic agitation, hirudin is dispersed in MES buffer, and adding mass concentration is 0.5% Tween 80.Salable freezing the saving backup of hirudin solution making.
(4) the hirudin solution of preparation in the sodium hyaluronate solution of preparation in step (2) and step (3) is evenly mixed.
In 2mL MES buffer, add the sodium hyaluronate solution 10mL making in step (2), the hirudin solution 4mL that then adds step (3) to make, the mass ratio of hirudin and hyaluronate sodium is 1:5, is uniformly mixed.
(5) in the mixed solution obtaining in step (4), add cross-linking agent.
Select 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC) solution and N-hydroxy-succinamide (NHS) solution as cross-linking agent, i.e. EDC/NHS cross-linking agent.Get the EDC solution 2mL of freshly prepared 50mg/ml, add in the mixed solution obtaining in step (4), hirudin and EDC mass ratio 1:5, after mix homogeneously standing 15 minutes.Then the NHS solution 2mL that gets freshly prepared 10mg/ml, adds described mixed solution, hirudin and NHS mass ratio 1:1.Under lucifuge condition, evenly mix, 12 as a child obtained hyaluronate sodium and hirudin crosslinking mixture, made described anticoagulant coatings material.
Described anticoagulant coatings material employing is soaked to Tu method and be coated on test material polyethylene (PE) sheet, polythene strip is done to infrared spectrum and detect, observe 1650-1750cm
-1and 1200-1320cm (C=O)
-1(C-N) amide (R1-CO-NH-R2) peak strengthens, and illustrates that hyaluronate sodium and hirudin generation amido link are crosslinked.
Described anticoagulant coatings material is carried out to anticoagulation test:
By described anticoagulant coatings material, adopt dip coating to be coated on polythene strip, polythene strip is cut into the square of 2cm*2cm, by centrifugal fresh blood collection platelet rich plasma, be inoculated on the polythene strip that has applied anticoagulant coatings, hatch after fixing dehydration, by sem observation platelet adhesion situation, by the hyaluronate sodium coating of unmodified hirudin as a control group, result shows, the unit are platelet adhesion quantity of the hyaluronate sodium coating of modifying hirudin be do not add the hyaluronate sodium coating of hirudin sample 30%, after covalent bond hirudin, improved the anticoagulation ability of coating.
Embodiment 5
A kind of anticoagulant coatings material, comprises hyaluronate sodium and hirudin, and described hyaluronate sodium forms gel network, and described hirudin is by amido link and described hyaluronate sodium covalent bond.The mass ratio of described hirudin and described hyaluronate sodium is 1:10.Described anticoagulant coatings material also comprises cross-linking agent, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC) and N-hydroxy-succinamide (NHS).
The preparation method of described anticoagulant coatings material, comprises the following steps:
(1) preparation 2-(N-morpholine) ethyl sulfonic acid (MES) buffer solution, concentration is 0.1mol/L, regulates pH value to 6.4.
(2) preparation sodium hyaluronate solution.
Get hyaluronate sodium (HA, molecular weight approximately 2,000,000) and, in 2-(N-morpholine) ethyl sulfonic acid buffer solution (MES, 0.1M, pH=6.4), it is fully dissolved and obtain sodium hyaluronate solution, sodium hyaluronate solution concentration is 20mg/ml.Can adopt magnetic agitation to accelerate hyaluronate sodium and dissolve, stir 12 hours.Salable freezing the saving backup of sodium hyaluronate solution making.
(3) preparation hirudin solution.
Get hirudin in MES buffer solution, hirudin concentration is 10mg/ml, by magnetic agitation, hirudin is dispersed in MES buffer, and adding mass concentration is 1.0% Tween 80.Salable freezing the saving backup of hirudin solution making.
(4) the hirudin solution of preparation in the sodium hyaluronate solution of preparation in step (2) and step (3) is evenly mixed.
In 4mL MES buffer, add the sodium hyaluronate solution 10mL making in step (2), the hirudin solution 2mL that then adds step (3) to make, the mass ratio of hirudin and hyaluronate sodium is 1:10, is uniformly mixed.
(5) in the mixed solution obtaining in step (4), add cross-linking agent.
Select 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC) solution and N-hydroxy-succinamide (NHS) solution as cross-linking agent, i.e. EDC/NHS cross-linking agent.Get the EDC solution 2mL of freshly prepared 50mg/ml, add in the mixed solution obtaining in step (4), hirudin and EDC mass ratio 1:5, after mix homogeneously standing 15 minutes.Then the NHS solution 2mL that gets freshly prepared 10mg/ml, adds described mixed solution, hirudin and NHS mass ratio 1:1.Under lucifuge condition, evenly mix, 12 as a child obtained hyaluronate sodium and hirudin crosslinking mixture, made described anticoagulant coatings material.
Described anticoagulant coatings material employing is soaked to Tu method and be coated on test material polyethylene (PE) sheet, polythene strip is done to infrared spectrum and detect, observe 1650-1750cm
-1and 1200-1320cm (C=O)
-1(C-N) amide (R1-CO-NH-R2) peak strengthens, and illustrates that hyaluronate sodium and hirudin generation amido link are crosslinked.
Described anticoagulant coatings material is carried out to anticoagulation test:
By described anticoagulant coatings material, adopt dip coating to be coated on polythene strip, polythene strip is cut into the square of 2cm*2cm, by centrifugal fresh blood collection platelet rich plasma, be inoculated on the polythene strip that has applied anticoagulant coatings, hatch after fixing dehydration, by sem observation platelet adhesion situation, by the hyaluronate sodium coating of unmodified hirudin as a control group, result shows, the unit are platelet adhesion quantity of the hyaluronate sodium coating of modifying hirudin be do not add the hyaluronate sodium coating of hirudin sample 50%, after covalent bond hirudin, improved the anticoagulation ability of coating.
Those skilled in the art will readily understand; the foregoing is only preferred embodiment of the present invention; not in order to limit the present invention, all any modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. an anticoagulant coatings material, is characterized in that, comprises hyaluronate sodium and hirudin, and described hyaluronate sodium forms gel network, and described hirudin is by amido link and described hyaluronate sodium covalent bond.
2. anticoagulant coatings material as claimed in claim 1, is characterized in that, the mass ratio of described hirudin and described hyaluronate sodium is 1:1 to 1:10.
3. anticoagulant coatings material as claimed in claim 2, is characterized in that, the mass ratio of described hirudin and described hyaluronate sodium is 1:3 to 1:5.
4. the anticoagulant coatings material as described in claims 1 to 3 any one, is characterized in that, described anticoagulant coatings material also comprises cross-linking agent.
5. anticoagulant coatings material as claimed in claim 4, is characterized in that, described cross-linking agent is 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides and N-hydroxy-succinamide.
6. a preparation method for anticoagulant coatings material, is characterized in that, comprises the following steps:
(1) preparation 2-(N-morpholine) ethyl sulfonic acid (MES) buffer solution.
(2) the MES buffer solution that uses step (1) to prepare, adds hyaluronate sodium, and concentration is 1mg/ml to 20mg/ml, fully dissolves and makes sodium hyaluronate solution;
(3) the MES buffer solution that uses step (1) to prepare, adds hirudin, and concentration is 1mg/ml to 10mg/ml, fully dissolves and makes hirudin solution;
(4) the hirudin solution of preparation in the sodium hyaluronate solution of preparation in step (2) and step (3) is evenly mixed, the mass ratio that makes hirudin and hyaluronate sodium is 1:1 to 1:10;
(5) in the mixed solution obtaining in step (4), add cross-linking agent, evenly mix, make hirudin and hyaluronate sodium covalent cross-linking, make anticoagulant coatings material.
7. anticoagulant coatings material preparation method as claimed in claim 6, is characterized in that, it is 0.1% to 1.0% Tween 80 that the hirudin solution in described step (3) contains mass concentration.
8. anticoagulant coatings material preparation method as claimed in claim 6, it is characterized in that, the cross-linking agent using in described step (5) is freshly prepared 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides solution and N-hydroxy-succinamide solution.
9. anticoagulant coatings material preparation method as claimed in claim 8, is characterized in that, the mass ratio of described hirudin, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides and N-hydroxy-succinamide is 1:5:1.
10. the anticoagulant coatings material preparation method as described in claim 6 to 9 any one, is characterized in that, also comprises:
(6) anticoagulant coatings material step (5) being made is dialysed, and removes cross-linking agent.
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