CN107519543A - With the fibrinolytic coating of fibrin ferment response and its application - Google Patents
With the fibrinolytic coating of fibrin ferment response and its application Download PDFInfo
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Abstract
The present invention relates to a kind of fibrinolytic coating with fibrin ferment response, including base material and modification, in the poly-dopamine adhesion layer of substrate surface, poly-dopamine adhesion layer is by amido link with having the fibrinolytic Nano capsule of fibrin ferment response to be connected.The invention also discloses application of the above-mentioned fibrinolytic coating with fibrin ferment response in antithrombotic apparatus is prepared.Fibrinolytic Nano capsule with fibrin ferment response is incorporated into face coat by the present invention, and the technology has application prospect in terms of human body is implanted into contacting blood material.
Description
Technical field
The present invention relates to bio-medical material and chemical field, more particularly to it is a kind of with fibrin ferment response
Fibrinolytic coating and its application.
Background technology
It is to cause human body to be implanted into one of the main reason for blood contacting devices application fails, mesh that material, which triggers the generation of thrombus,
The preceding main approach for solving the problem is to modify anti-thrombogenic coatings in material surface, and the main antithrombotic acitivity composition of coating is
Heparin or antiplatelet drug isoreactivity molecule.However, the Long Term Contact of antithrombotic acitivity molecule and blood will necessarily trigger blood
The dysfunction of liquid system, with serious complication, such as tissue bleeding.Preferable antithrombotic acitivity material should have thrombus
Response, i.e., only start antithrombotic mechanism when Coagulation test occurs, this also more meets the fortune of human body itself function point analysis mechanism
Operation mode.
Fibrin ferment is the distinctive products and major driver during Coagulation test, and its enzymolysis to fibrinogen is made
With the generation for promoting fibrin aggregation (nascent thrombus).In addition fibrin ferment can also feed back activation coagulation cascade process upstream
Clotting factor, cause the autoacceleration of Coagulation test.Fibrin ferment sound is introduced in the design of some antithrombotic reagents and pharmaceutical carrier
Performance is answered, allows medicament to discharge medicine at the position of thrombus generation.For example, pass through thrombin substrate polypeptide on thrombolytics surface
Albumin in grafting, so as to temporarily shelter its fibrinolytic, and position caused by fibrin ferment can by the enzymolysis of polypeptide and
Discharge active thrombolytics (Journal of Controlled Release, 177,42-50,2014).In another example utilize blood coagulation
The aptamer of enzyme be crosslinking points prepare microbubble as fibrin ferment response acoustic contrast agent (Biomaterials, 34,
9559-9565,2013)。
, can not practical application but the design of fibrin ferment response so far is only limitted to scale-model investigation.
The content of the invention
In order to solve the above technical problems, it is an object of the invention to provide a kind of fibrinolytic coating with fibrin ferment response and
It is applied, and the fibrinolytic Nano capsule with fibrin ferment response is incorporated into face coat by the present invention, and the technology is planted in human body
Have wide application prospects in terms of entering contacting blood material.
The invention provides a kind of fibrinolytic coating with fibrin ferment response, including base material and modification are in substrate surface
Poly-dopamine adhesion layer, poly-dopamine adhesion layer pass through amido link with fibrin ferment response fibrinolytic Nano capsule connect
Connect.For Nano capsule using hydrogel as shell, its surface is rich in amino, and poly-dopamine adhesion layer is rich in adjacent benzene hydroxyl and quinonyl, can be with
Michael's addition and schiff base reaction, therefore poly-dopamine adhesion layer and tool occurs with nucleophilic functional group such as amino or sulfydryl etc.
The fibrinolytic Nano capsule for having fibrin ferment response can form amido link by above-mentioned reaction, so as to connect.
Fibrinolytic Nano capsule with fibrin ferment response is core shell structure, and its center is Plasminogen
Agent, shell are the degradable hydrogel of fibrin ferment, and its preparation method comprises the following steps：
The polypeptide crosslinking agent that tissue-type plasminogen activator (t-PA), polymerized monomer and fibrin ferment can be cut off is being triggered
In the presence of agent and catalyst, Raolical polymerizable is carried out at 0-37 DEG C in pH=8-9 cushioning liquid, is had
The fibrinolytic Nano capsule of fibrin ferment response；Wherein, the cut-off polypeptide crosslinking agent of fibrin ferment can be cut off more including fibrin ferment
Peptide and its two terminal modified acrylamide groups, polymerized monomer are acrylamide and methacrylamide derivatives.
Further, methacrylamide derivatives are N- (3- aminopropyls) Methacrylamides or N- (3- diformazan ammonia
Base propyl group) Methacrylamide.
Further, the amino acid sequence of the cut-off polypeptide of fibrin ferment is as shown in SEQ ID No.1-SEQ ID No.4.
Xaa in SEQ ID No.2 represents Pip, and its full name is 4- amino piperidine -4- carboxylic acids, is in addition to 20 kinds of coded amino acids
A kind of special amino acid.
Preferably, the cut-off polypeptide crosslinking agent of fibrin ferment is：
Methacrylic acid-Gly-dPhe-Pro-Arg-Gly-Phe-Pro-Ala-Gly-Gly-Lys- methacrylic acids or
Acrylic acid-Gly-dPhe-Pro-Arg-Gly-Phe-Pro-Ala-Gly-Gly-Lys- acrylic acid, the methacrylic acid at both ends or
Acrylic acid is connected by amido link with polypeptide.
Further, the mol ratio of tissue-type plasminogen activator and polymerized monomer is 1:1500-6000；Fibrin ferment can
The polypeptide crosslinking agent of cut-out and the mol ratio of monomer are 1:4-16.
Further, the mol ratio of acrylamide and methacrylamide derivatives is 1-1.5:1.
Further, in reaction system, the concentration of catalyst is 10 μ g/mL-20 μ g/mL.
Further, reaction time 5-6h.
Further, initiator is water soluble, redox initiator ammonium persulfate or potassium peroxydisulfate；Catalyst is tetramethyl
Base ethylenediamine.
Further, cushioning liquid is sodium bicarbonate buffer liquid, PBS or Tri-HCl buffer solutions.
In the preparation method of above fibrinolytic Nano capsule, acrylamide passes through electrostatic masterpiece with methacrylamide derivatives
With the surface for adsorbing electronegative thrombolytic protein t-PA, and Raolical polymerizable in situ occurs on thrombolytic protein t-PA surfaces,
Thrombolysis activity molecule is wrapped up while hydrogel shell is formed under crosslinking agent existence condition, forms Nano capsule, the nanometre glue
The thrombolysis activity molecular core that capsule has the hydrogel shell of tridimensional network and is located therein.
Using the Nano capsule prepared by above method, the hydrogel shell of Nano capsule be can be cut off with fibrin ferment it is more
Formed after peptide cross-linking agents monomer, in the presence of fibrin ferment, fibrin ferment can cut off arginine-glycine in polypeptide
Connecting key, therefore hydrogel shell can degrade rapidly, be wrapped in t-PA therein and be released.T-PA rates of release with it is outer
The concentration of thrombin on boundary is directly proportional, is inversely proportional with the hydrogel shell degree of cross linking.Change the cut-off polypeptide crosslinking agent of fibrin ferment and
The mol ratio of monomer can change the degree of cross linking of hydrogel.
Further, poly-dopamine adhesion layer also molecule of the chemical bond connection with amino or sulfydryl, with amino or mercapto
One or more of the molecule of base in cysteine, polyethylene glycol amino, 11- sulfydryls undecylamine and chitosan.
Further, the preparation method of the fibrinolytic coating with fibrin ferment response comprises the following steps：
Be that solvent prepares dopamine solution by 8~9 buffer solution of pH, then by base material in dopamine solution 0~
6~24h is soaked at 37 DEG C, then is coated with the base material of dopamine in the fibrinolytic Nano capsule solution with fibrin ferment response
8~24h is soaked at 0~37 DEG C.Dopamine has in the presence of oxygen in alkaline aqueous solution in the generation of material surface
Autohemagglutination forms poly-dopamine film.
Above method can also comprise the following steps：By the base material after modification in the solution containing amino or the molecule of sulfydryl
Middle immersion.Amino or sulfydryl in molecule after adjacent benzene hydroxyl and the quinonyl effect on poly-dopamine surface with being modified base material table
Face.
Further, the molecule with amino or sulfydryl is selected from cysteine, polyethylene glycol amino (PEG-NH2), 11- mercaptos
One or more in base undecylamine and chitosan.Modification cysteine can make coating surface have antifouling property.
Further, the material of base material is dimethyl silicone polymer, polyurethane, stainless steel, gold, silicon rubber, latex, poly- third
Alkene, polyvinyl chloride, nylon, silicon, glass or titanium alloy.Base material is applied widely, can be that any poly-dopamine can glue
Attached base material.
The fibrinolytic coating with fibrin ferment response with chemically reactive poly-dopamine as adhesion layer by inciting somebody to action above
Fibrin ferment responsive nano capsule is carried on substrate surface and got, and the chemical property of poly-dopamine adhesion layer allows it to enter to advance
The modification of one step.The coating can only be degraded in the presence of fibrin ferment, and discharge thrombolysis molecule and play corroded rock mass, solidifying
In the case of hemase is present, t-PA is discharged with constant rate of speed, rate of release is directly proportional to concentration of thrombin existing for the external world；Work as the external world
In the presence of athrombia, coating stable, it will not degrade.
The invention also discloses application of the above-mentioned fibrinolytic coating with fibrin ferment response in antithrombotic apparatus is prepared.
In the presence of fibrin ferment, the fibrinolytic coating being dissolved in normal blood environment keeps inertia, and when thrombus generates and produces
During raw fibrin ferment, start fibrinolytic system, the t-PA that fibrinolytic coating is discharged can promote fibrinolytic to react, solution fibrin grumeleuse
(nascent thrombus).The antithrombotic equipment surfaces prepared based on the coating have the thrombolysis energy of fibrin ferment response (thrombus response)
Power, avoid traditional thrombolysis material and trigger a series of this drawback of complication due to the long-term exposure of bioactive molecule, in human body
There is application prospect in terms of implantation contacting blood material.
By such scheme, the present invention at least has advantages below：
1. the fibrinolytic coating of the present invention utilizes the fibrinolytic Nano capsule with fibrin ferment response to be made, the system of Nano capsule
Preparation Method is simple, smart structural design, using hydrogel as shell, parcel thrombolysis molecule, the polypeptide that can be cut off using fibrin ferment in it
As crosslinking agent, its response to fibrin ferment is realized.
2. the fibrinolytic coating of the present invention only just excites fibrinolytic function when Coagulation test occurs and produces fibrin ferment,
And it can keep stablizing inertia in normal blood environment.
3. for the fibrinolytic coating of the present invention using poly-dopamine as adhesion layer, applicable base materials are extensive.
Described above is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention,
And can be practiced according to the content of specification, below with presently preferred embodiments of the present invention and coordinate accompanying drawing describe in detail as after.
Brief description of the drawings
Fig. 1 is t- of the fibrinolytic coating of the preparation of the embodiment of the present invention 1 in the presence of having fibrin ferment and athrombia
PA release profiles；
Fig. 2 is that fibrinolytic coating prepared by the embodiment of the present invention 1 is molten in the presence of having fibrin ferment and athrombia
Bolt power curve；
Fig. 3 is table of the fibrinolytic coating of the preparation of the embodiment of the present invention 1 in the presence of having fibrin ferment and athrombia
Face thrombolysis molecular activity recovery curve；
Fig. 4 is activation recovering situation of the fibrinolytic coating of the preparation of the embodiment of the present invention 1 under various concentrations thrombin action.
Embodiment
With reference to the accompanying drawings and examples, the embodiment of the present invention is described in further detail.Implement below
Example is used to illustrate the present invention, but is not limited to the scope of the present invention.
Embodiment 1
It is 1mg/mL by tissue-type plasminogen activator's (t-PA) solution that 10 μ L concentration are 1mg/mL and 30 μ L concentration
Acrylamide solution and wait material amount N- (3- aminopropyls) methacrylamide hydrochloride solution mix
10min, adding the polypeptide that 2.5 μ L concentration are 50mg/mL, (sequence is acrylic acid-Gly-dPhe-Pro-Arg-Gly-Phe-Pro-
Ala-Gly-Gly-Lys (acrylic acid)) solution, 2.5 μ L concentration be 1mg/mL, 0.5 μ L N, N, N ', N '-tetramethylethylenediamine
(TEMED), and 200 μ L concentration be 0.1mg/mL ammonium persulfate solution (solution use pH=8.5 sodium bicarbonate buffer liquid
Prepare), carry out Raolical polymerizable at 25 DEG C, by gained mixed solution PBS 12h after 5h, that is, obtain blood coagulation
The Nano capsule solution of enzyme response corroded rock mass.
Dopamine solution using pH=8.5 three (methylol) aminomethane buffer solution compound concentrations as 5mg/mL, will be poly-
Urethane (PU) diaphragm is immersed in the dopamine solution, reacts 6h at 25 DEG C, and with PBS rinses three times, each rinse continues
10min, nitrogen drying surface, the PU pieces for being coated with dopamine are immersed in above-mentioned Nano capsule solution, reacted at 25 DEG C
12h, with PBS to the rinse of gained modified substrate surface three times, each rinse continue 10min, nitrogen drying surface.Again will
The modified surface of above-mentioned Nano capsule is dipped into the reduced glutathione solution that concentration is 10mg/mL and (uses 50mM, pH=
8.5 tris solution is prepared) in 24h, embathed three times, each 10min, that is, had repeatedly with ultra-pure water
The fibrinolytic coating of fibrin ferment response corroded rock mass.
The process that modified surface discharges t-PA in the presence of fibrin ferment is tested by isotope-labelling method.For conduct
Contrast, fibrinolytic coating is incubated jointly with buffer solution, as a result as shown in figure 1, t-PA can only in the presence of fibrin ferment
Significantly to discharge, and rate of release can be adjusted by changing concentration of thrombin.Solution after release is used for blood plasma thrombus
Dissolving test, as shown in Fig. 2 the fibrinolytic coating surface for occurring to degrade and discharge t-PA only under thrombin action can be
Dissolved again after thrombus generation.
By determining the situation of change of t-PA activity in nano surface capsule degradation process, as shown in figure 3, fibrinolytic coating table
Face can make nano surface capsule that shell degraded occur so that t-PA therein progressively recovers only in the presence of fibrin ferment
Enzymatic activity.Under special time, t-PA activation recovering degree is proportionate with concentration of thrombin in fibrinolytic coating, such as Fig. 4 institutes
Show.
Embodiment 2
It is 1mg/mL by tissue-type plasminogen activator's (t-PA) solution that 10 μ L concentration are 1mg/mL and 30 μ L concentration
Acrylamide solution and wait material amount N- (3- dimethylamino-propyls) methacryl amine aqueous solution mix 10min,
Adding the polypeptide that 2.5 μ L concentration are 50mg/mL, (sequence is methacrylic acid-Gly-dPhe-Pro-Arg-Gly-Phe-Pro-
Ala-Gly-Gly-Lys (methacrylic acid)) solution, 2.5 μ L concentration be 1mg/mL, 0.5 μ L N, N, N ', N '-tetramethyl second
Diamines (TEMED), and (solution is delayed the potassium persulfate solution that 200 μ L concentration are 0.1mg/mL with PH=8.5 sodium acid carbonate
Fliud flushing is prepared), carry out Raolical polymerizable at 25 DEG C, by gained mixed solution PBS 12h after 5h, that is, had
The Nano capsule solution of fibrin ferment response corroded rock mass.
Dopamine solution using pH=8.5 three (methylol) aminomethane buffer solution compound concentrations as 5mg/mL, will be poly-
Dimethyl siloxane (PDMS) diaphragm is immersed in the dopamine solution, reacts 6h at 25 DEG C, with PBS rinses three times, is moistened every time
Lasting 10min is washed, the PDMS pieces that dopamine is coated with behind nitrogen drying surface are immersed in above-mentioned Nano capsule solution, 25 DEG C
Lower reaction 12h, with PBS to the rinse of gained modified substrate surface three times, each rinse continue 10min, nitrogen drying table
Face.The modified surface of above-mentioned Nano capsule is dipped into the reduced glutathione solution that concentration is 10mg/mL again (to use
50mM, pH=8.5 tris solution is prepared) in 24h, embathed repeatedly with ultra-pure water three times, each 10min,
Obtain the fibrinolytic coating with fibrin ferment response corroded rock mass.
Embodiment 3
It is 1mg/mL by tissue-type plasminogen activator's (t-PA) solution that 10 μ L concentration are 1mg/mL and 30 μ L concentration
Acrylamide solution and wait material amount N- (3- aminopropyls) methacrylamide hydrochloride solution mix
10min, adding the polypeptide that 2.5 μ L concentration are 50mg/mL, (sequence is acrylic acid-Gly-dPhe-Pro-Arg-Gly-Phe-Pro-
Ala-Gly-Gly-Lys (acrylic acid)) solution, 2.5 μ L concentration be 1mg/mL, 0.5 μ L N, N, N ', N '-tetramethylethylenediamine
(TEMED), and 200 μ L concentration be 0.1mg/mL ammonium persulfate solution (solution use pH=8.5 sodium bicarbonate buffer liquid
Prepare), carry out Raolical polymerizable at 25 DEG C, by gained mixed solution PBS 12h after 5h, that is, obtain blood coagulation
The Nano capsule solution of enzyme response corroded rock mass.
, will not using pH=8.5 three (methylol) aminomethane buffer solution compound concentrations as 5mg/mL dopamine solution
Rust steel disc is immersed in the dopamine solution, reacts 6h at 25 DEG C, and with PBS rinses three times, each rinse continues 10min, nitrogen
Surface is dried up, the PU pieces for being coated with dopamine are immersed in above-mentioned Nano capsule solution, react 12h at 25 DEG C, buffered with PBS
To the rinse of gained modified substrate surface three times, each rinse continues 10min, nitrogen drying surface to liquid.Again by above-mentioned Nano capsule
Modified surface is dipped into the reduced glutathione solution that concentration is 10mg/mL and (uses 50mM, pH=8.5 trihydroxy methyl
Aminomethane solution prepare) in 24h, embathed three times, each 10min, that is, obtained with fibrin ferment response repeatedly with ultra-pure water
The fibrinolytic coating of corroded rock mass.
Embodiment 4
It is 2mg/mL by tissue-type plasminogen activator's (t-PA) solution that 10 μ L concentration are 1mg/mL and 30 μ L concentration
Acrylamide solution and wait material amount N- (3- aminopropyls) methacrylamide hydrochloride solution mix
10min, adding the polypeptide that 5 μ L concentration are 50mg/mL, (sequence is acrylic acid-Gly-dPhe-Pro-Arg-Gly-Phe-Pro-
Ala-Gly-Gly-Lys (acrylic acid)) solution, 2.5 μ L concentration be 1mg/mL, 0.5 μ L N, N, N ', N '-tetramethylethylenediamine
(TEMED), and 200 μ L concentration be 0.1mg/mL ammonium persulfate solution (solution use pH=8.5 sodium bicarbonate buffer liquid
Prepare), carry out Raolical polymerizable at 25 DEG C, by gained mixed solution PBS 12h after 5h, that is, obtain blood coagulation
The Nano capsule solution of enzyme response corroded rock mass.
Dopamine solution using pH=8.5 three (methylol) aminomethane buffer solution compound concentrations as 5mg/mL, will be poly-
Urethane (PU) diaphragm is immersed in the dopamine solution, reacts 6h at 25 DEG C, and with PBS rinses three times, each rinse continues
10min, nitrogen drying surface, the PU pieces for being coated with dopamine are immersed in above-mentioned Nano capsule solution, reacted at 25 DEG C
12h, with PBS to the rinse of gained modified substrate surface three times, each rinse continue 10min, nitrogen drying surface.Again will
The modified surface of above-mentioned Nano capsule is dipped into the reduced glutathione solution that concentration is 10mg/mL and (uses 50mM, pH=
8.5 tris solution is prepared) in 24h, embathed three times, each 10min, that is, had repeatedly with ultra-pure water
The fibrinolytic coating of fibrin ferment response corroded rock mass.
Embodiment 5
It is 1mg/mL by tissue-type plasminogen activator's (t-PA) solution that 10 μ L concentration are 1mg/mL and 30 μ L concentration
Acrylamide solution and wait material amount N- (3- aminopropyls) methacrylamide hydrochloride solution mix
10min, adding the polypeptide that 2.5 μ L concentration are 50mg/mL, (sequence is acrylic acid-Gly-dPhe-Pro-Arg-Gly-Phe-Pro-
Ala-Gly-Gly-Lys (acrylic acid)) solution, 2.5 μ L concentration be 1mg/mL, 0.5 μ L N, N, N ', N '-tetramethylethylenediamine
(TEMED), and 200 μ L concentration be 0.1mg/mL ammonium persulfate solution (solution use pH=8.5 sodium bicarbonate buffer liquid
Prepare), carry out Raolical polymerizable at 37 DEG C, by gained mixed solution PBS 12h after 5h, that is, obtain blood coagulation
The Nano capsule solution of enzyme response corroded rock mass.
Dopamine solution using pH=8.5 three (methylol) aminomethane buffer solution compound concentrations as 5mg/mL, will be poly-
Urethane (PU) diaphragm is immersed in the dopamine solution, reacts 6h at 25 DEG C, and with PBS rinses three times, each rinse continues
10min, nitrogen drying surface, the PU pieces for being coated with dopamine are immersed in above-mentioned Nano capsule solution, reacted at 25 DEG C
12h, with PBS to the rinse of gained modified substrate surface three times, each rinse continue 10min, nitrogen drying surface.Again will
The modified surface of above-mentioned Nano capsule is dipped into the reduced glutathione solution that concentration is 10mg/mL and (uses 50mM, pH=
8.5 tris solution is prepared) in 24h, embathed three times, each 10min, that is, had repeatedly with ultra-pure water
The fibrinolytic coating of fibrin ferment response corroded rock mass.
Embodiment 6
It is 1mg/mL by tissue-type plasminogen activator's (t-PA) solution that 10 μ L concentration are 1mg/mL and 30 μ L concentration
Acrylamide solution and wait material amount N- (3- aminopropyls) methacrylamide hydrochloride solution mix
10min, adding the polypeptide that 2.5 μ L concentration are 50mg/mL, (sequence is methacrylic acid-Gly-dPhe-Pro-Arg-Gly-Phe-
Pro-Ala-Gly-Gly-Lys (methacrylic acid)) solution, 2.5 μ L concentration be 1mg/mL, 0.5 μ L N, N, N ', N '-tetramethyl
Base ethylenediamine (TEMED), and (solution uses pH=8.5 bicarbonate to the ammonium persulfate solution that 200 μ L concentration are 0.1mg/mL
Sodium buffer), carry out Raolical polymerizable at 37 DEG C, by gained mixed solution PBS 12h after 5h, produce
To the Nano capsule solution for having fibrin ferment response corroded rock mass.
Dopamine solution using pH=8.5 three (methylol) aminomethane buffer solution compound concentrations as 5mg/mL, will be poly-
Urethane (PU) diaphragm is immersed in the dopamine solution, reacts 6h at 25 DEG C, and with PBS rinses three times, each rinse continues
10min, nitrogen drying surface, the PU pieces for being coated with dopamine are immersed in above-mentioned Nano capsule solution, reacted at 25 DEG C
12h, with PBS to the rinse of gained modified substrate surface three times, each rinse continue 10min, nitrogen drying surface.Again will
The modified surface of above-mentioned Nano capsule is dipped into the reduced glutathione solution that concentration is 10mg/mL and (uses 50mM, pH=
8.5 tris solution is prepared) in 24h, embathed three times, each 10min, that is, had repeatedly with ultra-pure water
The fibrinolytic coating of fibrin ferment response corroded rock mass.
Embodiment 7
It is 1mg/mL by tissue-type plasminogen activator's (t-PA) solution that 10 μ L concentration are 1mg/mL and 30 μ L concentration
Acrylamide solution and wait material amount N- (3- dimethylamino-propyls) methacryl amine aqueous solution mix 10min,
Adding the polypeptide that 2.5 μ L concentration are 50mg/mL, (sequence is methacrylic acid-Gly Gly Phe Pip Arg Ser Trp Gly
Cys Gly Lys- (methacrylic acid)) solution, 2.5 μ L concentration be 1mg/mL, 0.5 μ L N, N, N ', N '-tetramethyl second two
Amine (TEMED), and (solution uses PH=8.5 sodium bicarbonate buffer to the potassium persulfate solution that 200 μ L concentration are 0.1mg/mL
Liquid is prepared), carry out Raolical polymerizable at 25 DEG C, by gained mixed solution PBS 12h after 5h, that is, obtained solidifying
The Nano capsule solution of hemase response corroded rock mass.Wherein, the Pip full name in peptide sequence are 4- amino piperidine -4- carboxylic acids,
It is a kind of special amino acid in addition to 20 kinds of coded amino acids.
Dopamine solution using pH=8.5 three (methylol) aminomethane buffer solution compound concentrations as 5mg/mL, will be poly-
Dimethyl siloxane (PDMS) diaphragm is immersed in the dopamine solution, reacts 6h at 25 DEG C, with PBS rinses three times, is moistened every time
Lasting 10min is washed, the PDMS pieces that dopamine is coated with behind nitrogen drying surface are immersed in above-mentioned Nano capsule solution, 25 DEG C
Lower reaction 12h, with PBS to the rinse of gained modified substrate surface three times, each rinse continue 10min, nitrogen drying table
Face.The modified surface of above-mentioned Nano capsule is dipped into the reduced glutathione solution that concentration is 10mg/mL again (to use
50mM, pH=8.5 tris solution is prepared) in 24h, embathed repeatedly with ultra-pure water three times, each 10min,
Obtain the fibrinolytic coating with fibrin ferment response corroded rock mass.
Embodiment 8
It is 1mg/mL by tissue-type plasminogen activator's (t-PA) solution that 10 μ L concentration are 1mg/mL and 30 μ L concentration
Acrylamide solution and wait material amount N- (3- dimethylamino-propyls) methacryl amine aqueous solution mix 10min,
Adding the polypeptide that 2.5 μ L concentration are 50mg/mL, (sequence is acrylic acid-Gly Phe Pro Arg Gly Phe Pro Ala Gly
Gly Cys- (acrylic acid)) solution, 2.5 μ L concentration be 1mg/mL, 0.5 μ L N, N, N ', N '-tetramethylethylenediamine
(TEMED), and 200 μ L concentration be 0.1mg/mL potassium persulfate solution (solution use PH=8.5 sodium bicarbonate buffer liquid
Prepare), carry out Raolical polymerizable at 25 DEG C, by gained mixed solution PBS 12h after 5h, that is, obtain blood coagulation
The Nano capsule solution of enzyme response corroded rock mass.
Dopamine solution using pH=8.5 three (methylol) aminomethane buffer solution compound concentrations as 5mg/mL, will be poly-
Dimethyl siloxane (PDMS) diaphragm is immersed in the dopamine solution, reacts 6h at 25 DEG C, with PBS rinses three times, is moistened every time
Lasting 10min is washed, the PDMS pieces that dopamine is coated with behind nitrogen drying surface are immersed in above-mentioned Nano capsule solution, 25 DEG C
Lower reaction 12h, with PBS to the rinse of gained modified substrate surface three times, each rinse continue 10min, nitrogen drying table
Face.The modified surface of above-mentioned Nano capsule is dipped into the reduced glutathione solution that concentration is 10mg/mL again (to use
50mM, pH=8.5 tris solution is prepared) in 24h, embathed repeatedly with ultra-pure water three times, each 10min,
Obtain the fibrinolytic coating with fibrin ferment response corroded rock mass.
Described above is only the preferred embodiment of the present invention, is not intended to limit the invention, it is noted that for this skill
For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is some improvement and
Modification, these improvement and modification also should be regarded as protection scope of the present invention.
Sequence table
<110>University Of Suzhou
<120>With the fibrinolytic coating of fibrin ferment response and application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 11
<212> PRT
<213>Paramecium kind (Paramecium sp.)
<400> 1
Gly Phe Pro Arg Gly Phe Pro Ala Gly Gly Lys
1 5 10
<210> 2
<211> 10
<212> PRT
<213>Paramecium kind (Paramecium sp.)
<400> 2
Gly Gly Phe Xaa Arg Ser Trp Gly Cys Gly
1 5 10
<210> 3
<211> 11
<212> PRT
<213>Paramecium kind (Paramecium sp.)
<400> 3
Gly Phe Pro Arg Gly Phe Pro Ala Gly Gly Cys
1 5 10
<210> 4
<211> 10
<212> PRT
<213>Paramecium kind (Paramecium sp.)
<400> 4
Glu Leu Thr Pro Arg Gly Trp Arg Leu Glu
1 5 10
Claims (10)
- A kind of 1. fibrinolytic coating with fibrin ferment response, it is characterised in that：Including base material and modification in substrate surface Poly-dopamine adhesion layer, the poly-dopamine adhesion layer are connected by amido link and the fibrinolytic Nano capsule with fibrin ferment response Connect.
- 2. the fibrinolytic coating according to claim 1 with fibrin ferment response, it is characterised in that：It is described that there is fibrin ferment The fibrinolytic Nano capsule of response is core shell structure, and its center is tissue-type plasminogen activator, and shell is that fibrin ferment is degradable Hydrogel.
- 3. the fibrinolytic coating according to claim 1 with fibrin ferment response, it is characterised in that：It is described that there is fibrin ferment The preparation method of the fibrinolytic Nano capsule of response comprises the following steps：The polypeptide crosslinking agent that tissue-type plasminogen activator, polymerized monomer and fibrin ferment can be cut off is in initiator and catalyst In the presence of, Raolical polymerizable is carried out at 0-37 DEG C in pH=8-9 cushioning liquid, obtains described there is fibrin ferment The fibrinolytic Nano capsule of response；Wherein, the cut-off polypeptide crosslinking agent of the fibrin ferment includes the polypeptide that fibrin ferment can be cut off And its two terminal modified acrylamide groups, the polymerized monomer is acrylamide and methacrylamide derivatives.
- 4. the fibrinolytic coating according to claim 3 with fibrin ferment response, it is characterised in that：The methacryl Amine derivative is N- (3- aminopropyls) Methacrylamides or N- (3- dimethylamino-propyls) Methacrylamide.
- 5. the fibrinolytic coating according to claim 3 with fibrin ferment response, it is characterised in that：What fibrin ferment can be cut off The amino acid sequence of polypeptide is as shown in SEQ ID No.1-SEQ ID No.4.
- 6. the fibrinolytic coating according to claim 3 with fibrin ferment response, it is characterised in that：The tectotype fibrinolytic The mol ratio of zymoexcitator and polymerized monomer is 1:1500-6000；The fibrin ferment cut-off polypeptide crosslinking agent and monomer Mol ratio be 1:4-16.
- 7. the fibrinolytic coating according to claim 3 with fibrin ferment response, it is characterised in that：The acrylamide and The mol ratio of methacrylamide derivatives is 1-1.5:1.
- 8. the fibrinolytic coating according to claim 1 with fibrin ferment response, it is characterised in that：The poly-dopamine is glued Attached layer goes back molecule of the chemical bond connection with amino or sulfydryl, and the molecule with amino or sulfydryl is selected from cysteine, gathered One or more in ethylene glycol amino, 11- sulfydryls undecylamine and chitosan.
- 9. the fibrinolytic coating according to claim 1 with fibrin ferment response, it is characterised in that：The material of the base material For dimethyl silicone polymer, polyurethane, stainless steel, gold, silicon rubber, latex, polypropylene, polyvinyl chloride, nylon, silicon, glass or Titanium alloy.
- 10. the fibrinolytic coating with fibrin ferment response according to any one of claim 1-9 is preparing antithrombotic device Application in tool.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111072866A (en) * | 2019-12-27 | 2020-04-28 | 苏州大学 | High-tensile strong-adhesion photo-thermal hydrogel and preparation method and application thereof |
| CN111643726A (en) * | 2019-12-03 | 2020-09-11 | 东南大学 | Method for improving platelet activation resisting function of polyurethane material |
| CN113082300A (en) * | 2021-04-06 | 2021-07-09 | 西南交通大学 | Antibacterial and anticoagulant coating, preparation method and application thereof |
| WO2022021363A1 (en) * | 2020-07-31 | 2022-02-03 | 苏州大学 | Coating composition, hydrogel coating and preparation method therefor, and coated product |
| CN117660344A (en) * | 2023-08-11 | 2024-03-08 | 聊城市人民医院 | Double-network hydrogel surface and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104861177A (en) * | 2015-04-27 | 2015-08-26 | 苏州大学 | Water gel material with thrombin responsive thrombolysis capability and preparation method thereof |
| CN105778139A (en) * | 2016-03-04 | 2016-07-20 | 西北大学 | Method for constructing functional imitated cellulosa membrane stereo structure coating |
-
2017
- 2017-08-22 CN CN201710725300.XA patent/CN107519543B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104861177A (en) * | 2015-04-27 | 2015-08-26 | 苏州大学 | Water gel material with thrombin responsive thrombolysis capability and preparation method thereof |
| CN105778139A (en) * | 2016-03-04 | 2016-07-20 | 西北大学 | Method for constructing functional imitated cellulosa membrane stereo structure coating |
Non-Patent Citations (1)
| Title |
|---|
| 李聪等: "凝血酶响应性纤溶纳米胶囊的制备及性能研究", 《中国化学会2017全国高分子学术论文报告会摘要集——主题F：生物医用高分子》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111643726A (en) * | 2019-12-03 | 2020-09-11 | 东南大学 | Method for improving platelet activation resisting function of polyurethane material |
| CN111643726B (en) * | 2019-12-03 | 2022-03-29 | 东南大学 | Method for improving platelet activation resisting function of polyurethane material |
| CN111072866A (en) * | 2019-12-27 | 2020-04-28 | 苏州大学 | High-tensile strong-adhesion photo-thermal hydrogel and preparation method and application thereof |
| WO2022021363A1 (en) * | 2020-07-31 | 2022-02-03 | 苏州大学 | Coating composition, hydrogel coating and preparation method therefor, and coated product |
| CN113082300A (en) * | 2021-04-06 | 2021-07-09 | 西南交通大学 | Antibacterial and anticoagulant coating, preparation method and application thereof |
| CN117660344A (en) * | 2023-08-11 | 2024-03-08 | 聊城市人民医院 | Double-network hydrogel surface and preparation method and application thereof |
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