CN103721300B - Anticoagulation coating material and preparation method thereof - Google Patents
Anticoagulation coating material and preparation method thereof Download PDFInfo
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- CN103721300B CN103721300B CN201310700680.3A CN201310700680A CN103721300B CN 103721300 B CN103721300 B CN 103721300B CN 201310700680 A CN201310700680 A CN 201310700680A CN 103721300 B CN103721300 B CN 103721300B
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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, be widely used on the medical material that contacts with human blood and organizing, 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 of domestic and international medical circle concern.Anticoagulation bio-medical material has important using value.
Existing anticoagulant coatings at present, mainly adopts heparin as anticoagulin, heparin is fixed on material surface by physical absorption or chemically combined method.Adopt the method for 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, after covalentlying bind in metal support surface, its conformation is restricted, and anticoagulant active may be caused to reduce.Also there is biocompatibility issues in the coated polymeric in addition for fixing heparin, can not promote that endothelium heals, and also obviously do not contribute minimizing restenosis rate.
Also do not have at present a kind ofly to there is desirable blood coagulation resisting function and do not affect the anticoagulant coatings material of 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 to, by by anticoagulin hirudin stable to molecular weight, anticoagulant effect and the hyaluronic acid sodium gel network covalent bond with good biocompatibility, solve current anticoagulant coatings material anticoagulant effect technical problem that is unstable or permanent, poor biocompatibility thus.
For achieving the above object, according to one aspect of the present invention, provide a kind of anticoagulant coatings material, comprise 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, provide a kind of preparation method of anticoagulant coatings material, comprise the following steps:
(1) 2-(N-morpholine) ethyl sulfonic acid (MES) buffer solution (0.1mol/L, pH=6.4) is prepared.
(2) the MES buffer solution using step (1) to prepare, add hyaluronate sodium, concentration is 1mg/ml to 20mg/ml, fully dissolves obtained sodium hyaluronate solution;
(3) the MES buffer solution using step (1) to prepare, add hirudin, concentration is 1mg/ml to 10mg/ml, fully dissolves obtained hirudin solution;
(4) by the mixing of the hirudin dissolution homogeneity of preparation in the sodium hyaluronate solution of preparation in step (2) and step (3), the mass ratio of hirudin and hyaluronate sodium is made to be 1:1 to 1:10;
(5) add cross-linking agent, Homogeneous phase mixing in the mixed solution obtained in step (4), make hirudin and hyaluronate sodium covalent cross-linking, be i.e. obtained anticoagulant coatings material.
Preferably, described anticoagulant coatings material preparation method, the hirudin solution in its step (3) contains the Tween 80 that mass concentration is 0.1% to 1.0%.
Preferably, described anticoagulant coatings material preparation method, the cross-linking agent used 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) the anticoagulant coatings material that step (5) is obtained is dialysed, remove cross-linking agent.
In general, the above technical scheme conceived 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 of the direct Trombin inhibiting of energy is included in described anticoagulant coatings material as anticoagulant material.Hirudin causes thrombin to lose activity by the Binding Capacity group blocking thrombin, its free hydroxyl and thrombin free amino group are combined by electrostatic attraction, the complex produced can not dissociate within the scope of the whole pH that thrombin keeps active, and therefore hirudin has that anticoagulant active is high, the feature of Stability Analysis of Structures, non-easy in inactivation.Therefore described anticoagulant coatings material anticoagulant active is higher, and not easily because conformation change causes anticoagulin inactivation, anticoagulant effect is stablized.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 play significant anticoagulant effect, platelet adhesion reaction quantity drops to 500/mm
2below, as shown in Figure 1.Anticoagulant coatings material provided by the invention can play significant anticoagulant effect as can be seen here.
(2) because hirudin is small-molecular peptides, therefore independent as anticoagulant coatings material, run off and seriously cause anticoagulation time short.The present invention adopts stronger being cross-linked of viscosity to fix hirudin in netted hyaluronate sodium, and hirudin and hyaluronate sodium, by amido link covalent bond, form stable gel network, sustainable performance anticoagulant efficacy for a long time.
(3) hyaluronate sodium can provide high-hydrophilic top layer, be applicable to surface that is long-term and contacting blood, 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 have anticoagulant effect steady in a long-term, the use reducing anticoagulant can be helped, thus 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, amido link is adopted to make hirudin and hyaluronate sodium covalent bond, hirudin can be made to be combined with hyaluronate sodium stable, thus the anticoagulant effect making anticoagulant coatings materials serve permanent.There is no the method that bibliographical information can make to be formed between hirudin and hyaluronate sodium stable bond at present.
(5) due to anticoagulant coatings material preparation method provided by the invention, adopt cross-linking agent to help to form amido link, significantly can improve reaction efficiency, and selected cross-linking agent is removed 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 is gentle, is applicable to producing in enormous quantities.
Preferred version, optimize by doing the mass ratio range of hirudin and hyaluronate sodium, the kind of cross-linking agent and cross-linking agent adding proportion, make anticoagulant coatings material in anticoagulant effect, preparation difficulty, average out between production time and production cost, obtain cost comparatively cheap, produce relatively simple, 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 the electron scanning micrograph after hyaluronate sodium coating carries out platelet adhesion test.
Detailed description of the invention
In order to make object of the present invention, technical scheme and advantage clearly understand, 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, be not intended to limit the present invention.In addition, if below in described each embodiment of the present invention involved technical characteristic do not form conflict each other and just can mutually combine.
Hyaluronate sodium is the hyaluronic sodium salt of a kind of acid mucopolysaccharide.Hyaluronic acid is widely distributed in the various soft tissue of human body, gel can be formed, hyaluronic acid has the rheological properties of height viscoelasticity, permeability and uniqueness and good biocompatibility, and the gel of hyaluronic acid formation or a kind of absorbable biological material.
Hirudin is a kind of low molecular weight polypeptide be made up of 65 aminoacid, and the content of its Glutamic Acid and asparagine is higher, and molecular weight is about 7000, containing three disulfide bond.Hirudin is combined with the free amino group of thrombin by its free hydroxyl, thrombin is caused to lose activity, can not dissociate within the scope of the active pH of thrombin in this combination, therefore hirudin has the features such as anticoagulant active is high, Stability Analysis of Structures, non-easy in inactivation, in addition, hirudin later-period 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 that amido link is formed, 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) prepare 2-(N-morpholine) ethyl sulfonic acid (MES) buffer solution, concentration is 0.1mol/L, adjust ph to 6.4.
(2) sodium hyaluronate solution is prepared:
Get hyaluronate sodium (HA, molecular weight about 2,000,000) in 2-(N-morpholine) ethyl sulfonic acid buffer solution (MES, 0.1M, pH=6.4), to make it fully dissolve and obtain sodium hyaluronate solution, sodium hyaluronate solution concentration is 1mg/ml to 20mg/ml.Magnetic agitation can be adopted to accelerate hyaluronate sodium dissolve, stir 12 hours.The obtained salable freezen protective of sodium hyaluronate solution is for subsequent use.
(3) hirudin solution is prepared:
Get hirudin in MES buffer, hirudin is dispersed in MES buffer.For increasing the dissolubility of hirudin, can add surfactant, such as mass ratio is the Tween 80 of 0.1% to 1.0%.The obtained salable freezen protective of hirudin solution is for subsequent use.
(4) by the mixing of the hirudin dissolution homogeneity of preparation in the sodium hyaluronate solution of preparation in step (2) and step (3):
In MES buffer, add sodium hyaluronate solution obtained in step (2), then add hirudin solution, make the mass ratio of hirudin and hyaluronate sodium be 1:1 to 1:10, mix homogeneously.
(5) cross-linking agent is added in the mixed solution obtained 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 obtained in step (4), additional proportion is hirudin and EDC mass ratio 1:5, leaves standstill 15 minutes after mix homogeneously.Then get the NHS solution of freshly prepared 10mg/ml, add described mixed solution, additional proportion is hirudin and NHS mass ratio 1:1.Homogeneous phase mixing under lucifuge condition, 12 as a child obtained hyaluronate sodium and hirudin crosslinking mixture, i.e. obtained described anticoagulant coatings material.
(6) the anticoagulant coatings material that step (5) is obtained is dialysed, remove 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.Carry out platelet adhesion reaction test to anticoagulant coatings material provided by the invention, use Scanning Electron microscope imaging after test, 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, the significant anticoagulant effect of anticoagulant coatings materials serve provided by the invention.
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) prepare 2-(N-morpholine) ethyl sulfonic acid (MES) buffer solution, concentration is 0.1mol/L, adjust ph to 6.4.
(2) sodium hyaluronate solution is prepared.
Get hyaluronate sodium (HA, molecular weight about 2,000,000) in 2-(N-morpholine) ethyl sulfonic acid buffer solution (MES, 0.1M, pH=6.4), to make it fully dissolve and obtain sodium hyaluronate solution, sodium hyaluronate solution concentration is 1mg/ml.Magnetic agitation can be adopted to accelerate hyaluronate sodium dissolve, stir 12 hours.The obtained salable freezen protective of sodium hyaluronate solution is for subsequent use.
(3) hirudin solution is prepared.
Get hirudin in MES buffer, hirudin concentration is 1mg/ml, by magnetic agitation, hirudin is dispersed in MES buffer, adds the Tween 80 that mass concentration is 0.1%.The obtained salable freezen protective of hirudin solution is for subsequent use.
(4) by the mixing of the hirudin dissolution homogeneity of preparation in the sodium hyaluronate solution of preparation in step (2) and step (3).
In 6mL MES buffer, add sodium hyaluronate solution 20mL obtained in step (2), then add the hirudin solution 20mL that step (3) is obtained, the mass ratio of hyaluronate sodium and hirudin is 1:1, is uniformly mixed.
(5) cross-linking agent is added in the mixed solution obtained 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 2mL of freshly prepared 50mg/ml, add in the mixed solution obtained in step (4), hirudin and EDC mass ratio 1:5, after mix homogeneously, leave standstill 15 minutes.Then get the NHS solution 2mL of freshly prepared 10mg/ml, add described mixed solution, hirudin and NHS mass ratio 1:1.Homogeneous phase mixing under lucifuge condition, 12 as a child obtained hyaluronate sodium and hirudin crosslinking mixture, i.e. obtained described anticoagulant coatings material.
(6) the anticoagulant coatings material that step (5) is obtained is dialysed, remove cross-linking agent, thus purification anticoagulant coatings material.
Adopted by described anticoagulant coatings material leaching Tu method to be coated on test material polyethylene (PE) sheet, infrared spectrum is done to polythene strip and detects, 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 cross-linked.
Anticoagulation test is carried out to described anticoagulant coatings material:
By described anticoagulant coatings material, dip coating is adopted to be coated on polythene strip, polythene strip is cut into the square of 2cm*2cm, by fresh blood collected by centrifugation platelet rich plasma, be inoculated in and be coated with on the polythene strip of anticoagulant coatings, after hatching 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 is 45% of the sample of the hyaluronate sodium coating of not adding hirudin, the anticoagulation ability of coating is improve after covalent bond hirudin.
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) prepare 2-(N-morpholine) ethyl sulfonic acid (MES) buffer solution, concentration is 0.1mol/L, adjust ph to 6.4.
(2) sodium hyaluronate solution is prepared.
Get hyaluronate sodium (HA, molecular weight about 2,000,000) in 2-(N-morpholine) ethyl sulfonic acid buffer solution (MES, 0.1M, pH=6.4), to make it fully dissolve and obtain sodium hyaluronate solution, sodium hyaluronate solution concentration is 3mg/ml.Magnetic agitation can be adopted to accelerate hyaluronate sodium dissolve, stir 12 hours.The obtained salable freezen protective of sodium hyaluronate solution is for subsequent use.
(3) hirudin solution is prepared.
Get hirudin in MES buffer solution, hirudin concentration is 1mg/ml, by magnetic agitation, hirudin is dispersed in MES buffer, adds the Tween 80 that mass concentration is 0.1%.The obtained salable freezen protective of hirudin solution is for subsequent use.
(4) by the mixing of the hirudin dissolution homogeneity of preparation in the sodium hyaluronate solution of preparation in step (2) and step (3).
In 6mL MES buffer, add sodium hyaluronate solution 20mL obtained in step (2), then add the hirudin solution 20mL that step (3) is obtained, the mass ratio of hirudin and hyaluronate sodium is 1:3, is uniformly mixed.
(5) cross-linking agent is added in the mixed solution obtained 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 2mL of freshly prepared 50mg/ml, add in the mixed solution obtained in step (4), hirudin and EDC mass ratio 1:5, after mix homogeneously, leave standstill 15 minutes.Then get the NHS solution 2mL of freshly prepared 10mg/ml, add described mixed solution, hirudin and NHS mass ratio 1:1.Homogeneous phase mixing under lucifuge condition, 12 as a child obtained hyaluronate sodium and hirudin crosslinking mixture, i.e. obtained described anticoagulant coatings material.
Adopted by described anticoagulant coatings material leaching Tu method to be coated on test material polyethylene (PE) sheet, infrared spectrum is done to polythene strip and detects, 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 cross-linked.
Anticoagulation test is carried out to described anticoagulant coatings material:
By described anticoagulant coatings material, dip coating is adopted to be coated on polythene strip, polythene strip is cut into the square of 2cm*2cm, by fresh blood collected by centrifugation platelet rich plasma, be inoculated in and be coated with on the polythene strip of anticoagulant coatings, after hatching 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 is 23% of the sample of the hyaluronate sodium coating of not adding hirudin, the anticoagulation ability of coating is improve after covalent bond hirudin.
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) prepare 2-(N-morpholine) ethyl sulfonic acid (MES) buffer solution, concentration is 0.1mol/L, adjust ph to 6.4.
(2) sodium hyaluronate solution is prepared.
Get hyaluronate sodium (HA, molecular weight about 2,000,000) in 2-(N-morpholine) ethyl sulfonic acid buffer solution (MES, 0.1M, pH=6.4), to make it fully dissolve and obtain sodium hyaluronate solution, sodium hyaluronate solution concentration is 5mg/ml.Magnetic agitation can be adopted to accelerate hyaluronate sodium dissolve, stir 12 hours.The obtained salable freezen protective of sodium hyaluronate solution is for subsequent use.
(3) hirudin solution is prepared.
Get hirudin in MES buffer solution, hirudin concentration is 2.5mg/ml, by magnetic agitation, hirudin is dispersed in MES buffer, adds the Tween 80 that mass concentration is 0.5%.The obtained salable freezen protective of hirudin solution is for subsequent use.
(4) by the mixing of the hirudin dissolution homogeneity of preparation in the sodium hyaluronate solution of preparation in step (2) and step (3).
In 6mL MES buffer, add sodium hyaluronate solution 8mL obtained in step (2), then add the hirudin solution 4mL that step (3) is obtained, the mass ratio of hirudin and hyaluronate sodium is 1:4, is uniformly mixed.
(5) cross-linking agent is added in the mixed solution obtained 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 1mL of freshly prepared 50mg/ml, add in the mixed solution obtained in step (4), hirudin and EDC mass ratio 1:5, after mix homogeneously, leave standstill 15 minutes.Then get the NHS solution 1mL of freshly prepared 10mg/ml, add described mixed solution, hirudin and NHS mass ratio 1:1.Homogeneous phase mixing under lucifuge condition, 12 as a child obtained hyaluronate sodium and hirudin crosslinking mixture, i.e. obtained described anticoagulant coatings material.
(6) the anticoagulant coatings material that step (5) is obtained is dialysed, remove cross-linking agent, thus purification anticoagulant coatings material.
Adopted by described anticoagulant coatings material leaching Tu method to be coated on test material polyethylene (PE) sheet, infrared spectrum is done to polythene strip and detects, 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 cross-linked.
Anticoagulation test is carried out to described anticoagulant coatings material:
By described anticoagulant coatings material, dip coating is adopted to be coated on polythene strip, polythene strip is cut into the square of 2cm*2cm, by fresh blood collected by centrifugation platelet rich plasma, be inoculated in and be coated with on the polythene strip of anticoagulant coatings, after hatching 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 is 25% of the sample of the hyaluronate sodium coating of not adding hirudin, the anticoagulation ability of coating is improve after covalent bond hirudin.
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) prepare 2-(N-morpholine) ethyl sulfonic acid (MES) buffer solution, concentration is 0.1mol/L, adjust ph to 6.4.
(2) sodium hyaluronate solution is prepared:
Get hyaluronate sodium (HA, molecular weight about 2,000,000) in 2-(N-morpholine) ethyl sulfonic acid buffer solution (MES, 0.1M, pH=6.4), to make it fully dissolve and obtain sodium hyaluronate solution, sodium hyaluronate solution concentration is 10mg/ml.Magnetic agitation can be adopted to accelerate hyaluronate sodium dissolve, stir 12 hours.The obtained salable freezen protective of sodium hyaluronate solution is for subsequent use.
(3) hirudin solution is prepared.
Get hirudin in MES buffer solution, hirudin concentration is 5mg/ml, by magnetic agitation, hirudin is dispersed in MES buffer, adds the Tween 80 that mass concentration is 0.5%.The obtained salable freezen protective of hirudin solution is for subsequent use.
(4) by the mixing of the hirudin dissolution homogeneity of preparation in the sodium hyaluronate solution of preparation in step (2) and step (3).
In 2mL MES buffer, add sodium hyaluronate solution 10mL obtained in step (2), then add the hirudin solution 4mL that step (3) is obtained, the mass ratio of hirudin and hyaluronate sodium is 1:5, is uniformly mixed.
(5) cross-linking agent is added in the mixed solution obtained 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 2mL of freshly prepared 50mg/ml, add in the mixed solution obtained in step (4), hirudin and EDC mass ratio 1:5, after mix homogeneously, leave standstill 15 minutes.Then get the NHS solution 2mL of freshly prepared 10mg/ml, add described mixed solution, hirudin and NHS mass ratio 1:1.Homogeneous phase mixing under lucifuge condition, 12 as a child obtained hyaluronate sodium and hirudin crosslinking mixture, i.e. obtained described anticoagulant coatings material.
Adopted by described anticoagulant coatings material leaching Tu method to be coated on test material polyethylene (PE) sheet, infrared spectrum is done to polythene strip and detects, 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 cross-linked.
Anticoagulation test is carried out to described anticoagulant coatings material:
By described anticoagulant coatings material, dip coating is adopted to be coated on polythene strip, polythene strip is cut into the square of 2cm*2cm, by fresh blood collected by centrifugation platelet rich plasma, be inoculated in and be coated with on the polythene strip of anticoagulant coatings, after hatching 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 is 30% of the sample of the hyaluronate sodium coating of not adding hirudin, the anticoagulation ability of coating is improve after covalent bond hirudin.
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) prepare 2-(N-morpholine) ethyl sulfonic acid (MES) buffer solution, concentration is 0.1mol/L, adjust ph to 6.4.
(2) sodium hyaluronate solution is prepared.
Get hyaluronate sodium (HA, molecular weight about 2,000,000) in 2-(N-morpholine) ethyl sulfonic acid buffer solution (MES, 0.1M, pH=6.4), to make it fully dissolve and obtain sodium hyaluronate solution, sodium hyaluronate solution concentration is 20mg/ml.Magnetic agitation can be adopted to accelerate hyaluronate sodium dissolve, stir 12 hours.The obtained salable freezen protective of sodium hyaluronate solution is for subsequent use.
(3) hirudin solution is prepared.
Get hirudin in MES buffer solution, hirudin concentration is 10mg/ml, by magnetic agitation, hirudin is dispersed in MES buffer, adds the Tween 80 that mass concentration is 1.0%.The obtained salable freezen protective of hirudin solution is for subsequent use.
(4) by the mixing of the hirudin dissolution homogeneity of preparation in the sodium hyaluronate solution of preparation in step (2) and step (3).
In 4mL MES buffer, add sodium hyaluronate solution 10mL obtained in step (2), then add the hirudin solution 2mL that step (3) is obtained, the mass ratio of hirudin and hyaluronate sodium is 1:10, is uniformly mixed.
(5) cross-linking agent is added in the mixed solution obtained 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 2mL of freshly prepared 50mg/ml, add in the mixed solution obtained in step (4), hirudin and EDC mass ratio 1:5, after mix homogeneously, leave standstill 15 minutes.Then get the NHS solution 2mL of freshly prepared 10mg/ml, add described mixed solution, hirudin and NHS mass ratio 1:1.Homogeneous phase mixing under lucifuge condition, 12 as a child obtained hyaluronate sodium and hirudin crosslinking mixture, i.e. obtained described anticoagulant coatings material.
Adopted by described anticoagulant coatings material leaching Tu method to be coated on test material polyethylene (PE) sheet, infrared spectrum is done to polythene strip and detects, 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 cross-linked.
Anticoagulation test is carried out to described anticoagulant coatings material:
By described anticoagulant coatings material, dip coating is adopted to be coated on polythene strip, polythene strip is cut into the square of 2cm*2cm, by fresh blood collected by centrifugation platelet rich plasma, be inoculated in and be coated with on the polythene strip of anticoagulant coatings, after hatching 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 is 50% of the sample of the hyaluronate sodium coating of not adding hirudin, the anticoagulation ability of coating is improve after covalent bond hirudin.
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 amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. an anticoagulant coatings material, it is characterized in that, comprise hyaluronate sodium and hirudin, described hyaluronate sodium forms gel network, described hirudin is by amido link and described hyaluronate sodium covalent bond, and the mass ratio of described hirudin and described hyaluronate sodium is 1:1 to 1:10.
2. anticoagulant coatings material as claimed in claim 1, it is characterized in that, the mass ratio of described hirudin and described hyaluronate sodium is 1:3 to 1:5.
3. anticoagulant coatings material as claimed in claim 1 or 2, it is characterized in that, described anticoagulant coatings material also comprises cross-linking agent.
4. anticoagulant coatings material as claimed in claim 3, it is characterized in that, described cross-linking agent is 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides and N-hydroxy-succinamide.
5. a preparation method for anticoagulant coatings material, is characterized in that, comprises the following steps:
(1) 2-(N-morpholine) ethyl sulfonic acid (MES) buffer solution is prepared.
(2) the MES buffer solution using step (1) to prepare, add hyaluronate sodium, concentration is 1mg/ml to 20mg/ml, fully dissolves obtained sodium hyaluronate solution;
(3) the MES buffer solution using step (1) to prepare, add hirudin, concentration is 1mg/ml to 10mg/ml, fully dissolves obtained hirudin solution;
(4) by the mixing of the hirudin dissolution homogeneity of preparation in the sodium hyaluronate solution of preparation in step (2) and step (3), the mass ratio of hirudin and hyaluronate sodium is made to be 1:1 to 1:10;
(5) add cross-linking agent, Homogeneous phase mixing in the mixed solution obtained in step (4), make hirudin and hyaluronate sodium covalent cross-linking, be i.e. obtained anticoagulant coatings material.
6. anticoagulant coatings material preparation method as claimed in claim 5, it is characterized in that, the hirudin solution in described step (3) contains the Tween 80 that mass concentration is 0.1% to 1.0%.
7. anticoagulant coatings material preparation method as claimed in claim 5, it is characterized in that, the cross-linking agent used in described step (5) is freshly prepared 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides solution and N-hydroxy-succinamide solution.
8. anticoagulant coatings material preparation method as claimed in claim 7, it 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.
9. the anticoagulant coatings material preparation method as described in claim 5 to 8 any one, is characterized in that, also comprise:
(6) the anticoagulant coatings material that step (5) is obtained is dialysed, remove cross-linking agent.
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| CN106730051B (en) * | 2016-12-27 | 2020-05-01 | 生纳科技(上海)有限公司 | Anticoagulation polymer biological material and preparation method and application thereof |
| CN107456611A (en) * | 2017-07-23 | 2017-12-12 | 北京化工大学 | A kind of preparation method of anticoagulation composite coating |
| CN111840664A (en) * | 2020-08-11 | 2020-10-30 | 山东朱氏药业集团有限公司 | Medical material and method for preparing anticoagulant coating on surface of medical material |
| CN114213905A (en) * | 2021-12-24 | 2022-03-22 | 李金彪 | Dialysis harbor and dialysis harbor surface coating material and preparation method thereof |
| CN115109293B (en) * | 2022-07-26 | 2023-10-27 | 浙江硕华生命科学研究股份有限公司 | Blood sedimentation test tube and preparation method thereof |
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