CN113817048A - Anticoagulation titanium-based material and construction method thereof - Google Patents
Anticoagulation titanium-based material and construction method thereof Download PDFInfo
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- CN113817048A CN113817048A CN202110939931.8A CN202110939931A CN113817048A CN 113817048 A CN113817048 A CN 113817048A CN 202110939931 A CN202110939931 A CN 202110939931A CN 113817048 A CN113817048 A CN 113817048A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/02—Inorganic materials
- A61L31/022—Metals or alloys
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/08—Materials for coatings
- A61L31/10—Macromolecular materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L31/16—Biologically active materials, e.g. therapeutic substances
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
- A61L33/0005—Use of materials characterised by their function or physical properties
- A61L33/0011—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate
- A61L33/0041—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate characterised by the choice of an antithrombatic agent other than heparin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
- A61L33/02—Use of inorganic materials
- A61L33/022—Metal or alloys
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
- A61L33/06—Use of macromolecular materials
- A61L33/12—Polypeptides, proteins or derivatives thereof, e.g. degradation products thereof
- A61L33/128—Other specific proteins or polypeptides not covered by A61L33/122 - A61L33/126
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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- C—CHEMISTRY; METALLURGY
- C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
- C23C—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; SURFACE TREATMENT OF METALLIC MATERIAL BY DIFFUSION INTO THE SURFACE, BY CHEMICAL CONVERSION OR SUBSTITUTION; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL
- C23C26/00—Coating not provided for in groups C23C2/00 - C23C24/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/42—Anti-thrombotic agents, anticoagulants, anti-platelet agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
- A61L2300/606—Coatings
Abstract
The invention discloses an anticoagulant material and a preparation method thereof, wherein a medical pure titanium material is used as a substrate, a polypeptide sequence with a specific binding site is screened and verified, the polypeptide sequence is combined with a recombinant hirudin polypeptide molecule, and the anticoagulant titanium-based material is constructed by a controllable self-assembly technology. The preparation method of the anticoagulation titanium-based material is simple and easy to implement, and can remarkably improve the protein adhesion resistance and anticoagulation performance of the surface of the titanium-based cardiovascular stent material; meanwhile, the surface of the prepared material has excellent blood compatibility and cell compatibility, shows low hemolysis rate and can obviously promote endothelialization.
Description
Technical Field
The invention belongs to the technical field of biomedical treatment, and particularly relates to an anticoagulation titanium-based material and a construction method thereof.
Background
Recombinant Hirudin (Hirudin) is a low-molecular protein extracted from leeches and salivary glands thereof, has an active site capable of specifically recognizing thrombin at the N end and the C end of a molecular structure, is tightly combined with thrombin according to a ratio of 1:1 to form a compound, plays an anticoagulant role, and is widely concerned by researchers.
The titanium-based material has excellent mechanical properties and cell compatibility, so that the titanium-based material is widely applied to orthopedics and cardiovascular departments clinically and becomes an important biological material. However, when the titanium-based material is used as a cardiovascular stent material, the problems of protein adhesion, blood coagulation, thrombus and the like are very easy to occur due to poor surface blood compatibility, and the life safety of a patient is seriously threatened. Therefore, the method has significant research significance on how to improve the protein adhesion resistance and the anticoagulation performance of the surface of the titanium-based cardiovascular stent material, reduce the risk of thrombus and keep excellent cell compatibility.
Disclosure of Invention
The invention aims to overcome the technical defects that the surface of the existing titanium-based material has poor blood compatibility, is very easy to generate protein adhesion and blood coagulation phenomena and causes thrombus, and provides a method for constructing the titanium-based material with the anticoagulation function.
The technical scheme adopted by the invention is as follows:
in a first aspect of the invention, there is provided a recombinant hirudin polypeptide comprising at least one of the following (I) to (IV):
(I) comprises at least one peptide fragment of VYTDCTESGLCCEGSHVCGQGNKCILGSDGEKNOCCETTCGTPKPQSHNDGDFEEIPEEYLO;
(ii) comprises at least one SKHKGGKHKGGKHKGKSSGK peptide stretch;
(iii) comprises at least one SKHKGGKHKGGKHKG peptide linked to a KSSGK peptide;
(IV) comprises at least one SKHKGGKHKGGKHKG peptide stretch linked to a tyrosine residue; at least one of the tyrosine residues is linked to a KSSGK peptide fragment.
Preferably, the polypeptide comprises at least one of the following (i) to (IV):
(I) comprises a peptide fragment of VYTDCTESGLCCEGSHVCGQGNKCILGSDGEKNOCCETTCPKPQSHNDGDFEEIPEEYLO;
comprises an SKHKGGKHKGGKHKGKSSGK peptide segment;
(II) comprising two SKHKGGKHKGGKHKG peptides linked to a KSSGK peptide;
(iii) comprises two SKHKGGKHKGGKHKG peptide fragments linked to a single tyrosine residue; two of the described tyrosine residues are attached to one KSSGK peptide fragment.
More preferably, the recombinant hirudin polypeptide has the structure shown in any one of formulas (I) to (IV):
(IV). In some embodiments of the present application, the polypeptides are represented by the structures shown in FIGS. 1 to 4.
Note that O is pyrrolysine Pyl.
In a second aspect of the invention, there is provided a nucleic acid molecule encoding a polypeptide according to the first aspect of the invention.
In a third aspect of the invention, there is provided the use of a polypeptide according to the first aspect of the invention or a nucleic acid molecule according to the second aspect of the invention in the preparation of an anticoagulant.
In a fourth aspect of the present invention, there is provided an anticoagulant material, wherein the surface of the substrate material is coated with the polypeptide of the first aspect of the present invention.
According to the fourth aspect of the invention, preferably the recombinant hirudin polypeptide contains an amino acid group which specifically binds to the titanium hydroxyl group.
Preferably, the recombinant hirudin polypeptide is attached to the surface of the substrate material via an amino-terminal sequence.
According to the fourth aspect of the present invention, preferably, the base material is pure titanium.
In a fifth aspect of the present invention, there is provided a method for preparing the anticoagulant material according to the fourth aspect of the present invention, comprising the following steps:
s1, carrying out acid treatment on a medical pure titanium material;
s2, carrying out plasma treatment on the acid-treated pure titanium material;
s3, immersing the pure titanium material treated in the step S2 into a solution containing the recombinant hirudin polypeptide to perform self-assembly reaction;
and S4, cleaning to obtain the titanium-based cardiovascular stent.
Further, according to the preparation method of the fifth aspect of the present invention, the acid treatment in step S1 is hydrofluoric acid treatment, the concentration of hydrofluoric acid is preferably 5% to 10%, and the treatment time is preferably 1 to 3 minutes.
Further, according to the preparation method of the fifth aspect of the present invention, the atmosphere of the plasma treatment in the step S2 is oxygen or air, and the treatment time is preferably 1 to 10 min.
Further, according to the preparation method of the fifth aspect of the present invention, the solvent of the solution containing the recombinant hirudin polypeptide in step S3 is a mixture of water and ethanol, wherein the volume ratio of ethanol is 0% to 100%;
the concentration of the solution containing the recombinant hirudin polypeptide is preferably 0.01-10 mM;
the reaction time of the assembly reaction is preferably 20-720 min, and the reaction temperature is preferably 25-60 ℃.
Further, according to the preparation method of the fifth aspect of the present invention, the cleaning method in step S4 is ultrasonic cleaning with ethanol and deionized water, respectively, and preferably, the cleaning time is about 5 min.
In a sixth aspect of the present invention, the anticoagulant material according to the fourth aspect of the present invention is used for preparing a cardiovascular stent.
The invention has the beneficial effects that:
the invention provides a recombinant hirudin polypeptide molecule which has a site specifically combined with a titanium-based material and can be used for preparing an anticoagulant material.
The invention provides an anticoagulant material, which is prepared by taking a medical pure titanium material as a substrate, combining the substrate with recombinant hirudin polypeptide molecules and constructing the anticoagulant material on the surface of the titanium-based material through a controllable self-assembly technology, so that the anticoagulant material has a good anticoagulant function and can be used as a cardiovascular stent.
The invention also provides a preparation method of the anticoagulation titanium-based material, which utilizes the charge interaction between the positive charge of the KHK sequence in the modified recombinant hirudin polypeptide and the titanium hydroxyl on the surface of the titanium substrate after plasma treatment; the recombinant hirudin polypeptide is covered on the surface of the titanium material, the method is simple and easy to implement, and the protein adhesion resistance and the anticoagulation performance of the surface of the titanium-based cardiovascular stent material can be obviously improved; meanwhile, the surface of the prepared material has excellent blood compatibility and cell compatibility, shows low hemolysis rate and can obviously promote endothelialization.
Drawings
FIG. 1 shows the original amino acid sequence of hirudin.
FIG. 2 shows the sequence of modified recombinant hirudin polypeptide molecule 1.
FIG. 3 shows the sequence of modified recombinant hirudin polypeptide molecule 2.
FIG. 4 shows the sequence of modified recombinant hirudin polypeptide molecule 3.
FIG. 5 shows the results of the anti-protein adhesion and anti-platelet adhesion of the recombinant hirudin polypeptide anticoagulant material.
FIG. 6 shows the results of the hemolysis rate of the recombinant hirudin polypeptide anticoagulant material.
FIG. 7 shows the result of the cell compatibility of the anticoagulant material of recombinant hirudin polypeptide to vascular endothelial cells.
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.
(1) Anti-protein adhesion assay
Human serum albumin and fibronectin were dissolved in PBS solution to prepare 0.2mg/mL solutions, respectively. The sample was placed in a QCM-D instrument, the peristaltic pump was turned on, and the baseline was leveled with PBS buffer. And then respectively introducing the protein solution, and after the adsorption curve is stable, introducing PBS again to stabilize the curve. In the experiment process, the natural frequency of the QCM-D is 4.95MHz, and the vibration frequency and the dissipation factor of each sample are detected, so that the surface protein adsorption resistance is calculated.
(2) Anti-platelet adhesion test
The samples were placed in 0.1mL platelet rich plasma, incubated at 37 ℃ for 2h, and then the surface was gently rinsed with PBS. The sample is placed in 2% glutaraldehyde fixing solution for fixing for 4h, and is dehydrated by using gradient ethanol (30% -100%). And finally, soaking the blood cells for 30min by using isoamyl acetate, carrying out electron microscope test after air drying, and counting the number of the blood platelets.
(3) Hemolysis test
This patent adopts rabbit erythrocyte to evaluate the hemolytic activity on sample surface. The samples were incubated in 500. mu.L of 5% red blood cell solution (in PBS). And 0.1% Triton X-100 and PBS incubated red blood cells were used as positive and negative controls, respectively. After 3h incubation, the red blood cell solution was collected and centrifuged at 800g for 10 min. The hemolysis rate was calculated by taking 100. mu.L of the supernatant and placing it in a 96-well plate and testing it for OD absorbance at 540 nm.
(4) Cell compatibility test of vascular endothelial cells
Vascular endothelial cells are cultured on the surface of the sample, and after 24 hours, the sample is taken out and the cell activity on the surface without the sample is tested by using a CCK8 method.
Control group: an unmodified pure titanium material was used as a control.
There is provided a recombinant hirudin polypeptide comprising at least one of the following (i) to (IV):
(I) comprises at least one peptide fragment of VYTDCTESGLCCEGSHVCGQGNKCILGSDGEKNOCCETTCGTPKPQSHNDGDFEEIPEEYLO (SEQ ID NO. 1);
(II) comprises at least one SKHKGGKHKGGKHKGKSSGK (SEQ ID NO.2) peptide fragment;
(III) comprising at least one SKHKGGKHKGGKHKG (SEQ ID NO.3) peptide linked to a KSSGK (SEQ ID NO.4) peptide;
(IV) comprises at least one SKHKGGKHKGGKHKG (SEQ ID No.3) peptide stretch linked to a tyrosine residue; at least one of the said tyrosine residues is linked to a KSSGK (SEQ ID NO.4) peptide stretch.
The original sequence of hirudin is shown in FIG. 1. Then, the hirudin original sequence is connected with the KSSGK peptide segment or is further connected with a tyrosine residue and then is connected with SKHKGGKHKGGKHKG peptide segments to construct 3 new recombinant hirudin polypeptides, the structures of which are shown in formulas (II) to (IV) (corresponding to the attached figures 2 to 4):
example 1
The preparation method of the anticoagulant material comprises the following steps:
(1) carrying out hydrofluoric acid treatment on the medical pure titanium material, wherein the concentration of hydrofluoric acid is 10%, and the treatment time is 3 min;
(2) carrying out plasma treatment on the sample subjected to acid treatment, wherein the atmosphere of the plasma treatment is oxygen, and the treatment time is 10 min;
(3) immersing the sample treated in the steps (1) and (2) into a modified recombinant hirudin solution for reaction, and carrying out self-assembly reaction; wherein the structure of the modified recombinant hirudin is shown in figure 2; the solvent of the modified recombinant hirudin solution is 80% ethanol water solution; the concentration of the modified recombinant hirudin solution is 10 mM; the reaction time is 720min, and the reaction temperature is 60 ℃;
(4) after the reaction, the sample is ultrasonically cleaned for 5min by using ethanol and deionized water respectively to obtain the anticoagulation titanium-based material.
Anti-protein adhesion experiments show that compared with an unmodified control group, the constructed material can inhibit the adhesion of 94.8% of human serum albumin and 92.3% of fibronectin; the results are shown in FIG. 5.
An anti-platelet adhesion experiment shows that compared with an unmodified control group, the constructed material can inhibit the platelet adhesion by 98.2 percent; the results are shown in FIG. 5.
The hemolysis experiment result shows that compared with a positive control group, the hemolysis rate of the constructed material is 0.4%; the results are shown in FIG. 6. The cell compatibility experiment result of the vascular endothelial cells shows that the activity of the surface cells of the constructed material is improved by 13.2 percent compared with that of an unmodified control group within 24 hours; the results are shown in FIG. 7.
Example 2
The preparation method of the anticoagulant material comprises the following steps:
(1) carrying out hydrofluoric acid treatment on the medical pure titanium material, wherein the concentration of hydrofluoric acid is 5%, and the treatment time is 1 min;
(2) carrying out plasma treatment on the sample subjected to acid treatment, wherein the atmosphere of the plasma treatment is air, and the treatment time is 1 min;
(3) immersing the sample treated in the steps (1) and (2) into a modified recombinant hirudin solution for reaction, and carrying out self-assembly reaction; wherein the structure of the modified recombinant hirudin is shown in figure 3; the solvent of the modified recombinant hirudin solution is 100% ethanol solution; the concentration of the modified recombinant hirudin solution is 0.01 mM; the reaction time is 20min, and the reaction temperature is 25 ℃;
(4) after the reaction, the sample is ultrasonically cleaned for 5min by using ethanol and deionized water respectively to obtain the anticoagulation titanium-based material.
Anti-protein adhesion experiments showed that the constructed material was able to inhibit the adhesion of 53.2% human serum albumin and 44.1% fibronectin compared to the unmodified control;
an anti-platelet adhesion experiment shows that compared with an unmodified control group, the constructed material can inhibit 20.3% of platelet adhesion;
the hemolysis experiment result shows that compared with a positive control group, the hemolysis rate of the constructed material is-0.2%;
the cell compatibility experiment result of the vascular endothelial cells shows that the cell activity of the surface of the constructed material is improved by 1.3 percent compared with that of an unmodified control group within 24 hours.
Example 3
The preparation method of the anticoagulant material comprises the following steps:
(1) carrying out hydrofluoric acid treatment on the medical pure titanium material, wherein the concentration of hydrofluoric acid is 10%, and the treatment time is 3 min;
(2) carrying out plasma treatment on the sample subjected to acid treatment, wherein the atmosphere of the plasma treatment is oxygen, and the treatment time is 8 min;
(3) immersing the sample treated in the steps (1) and (2) into a modified recombinant hirudin solution for reaction, and carrying out self-assembly reaction; wherein the structure of the modified recombinant hirudin is shown in figure 4; the solvent of the modified recombinant hirudin solution is 90% ethanol water solution; the concentration of the modified recombinant hirudin solution is 10 mM; the reaction time is 720min, and the reaction temperature is 60 ℃;
(4) after the reaction, the sample is ultrasonically cleaned for 5min by using ethanol and deionized water respectively to obtain the titanium-based cardiovascular stent material with the anticoagulation function.
Anti-protein adhesion experiments show that compared with an unmodified control group, the constructed material can inhibit the adhesion of 95.3% of human serum albumin and 94.8% of fibronectin;
an anti-platelet adhesion experiment shows that compared with an unmodified control group, the constructed material can inhibit 99.2% of platelet adhesion;
the hemolysis experiment result shows that compared with the positive control group, the hemolysis rate of the constructed material is 0.8%;
the cell compatibility experiment result of the vascular endothelial cells shows that the cell activity of the surface of the constructed material is improved by 15.3 percent compared with that of an unmodified control group within 24 hours.
Example 4
The preparation method of the anticoagulant material comprises the following steps:
(1) carrying out hydrofluoric acid treatment on the medical pure titanium material, wherein the concentration of hydrofluoric acid is 8%, and the treatment time is 2 min;
(2) carrying out plasma treatment on the sample subjected to acid treatment, wherein the atmosphere of the plasma treatment is oxygen, and the treatment time is 8 min;
(3) immersing the sample treated in the steps (1) and (2) into a modified recombinant hirudin solution for reaction, and carrying out self-assembly reaction; wherein the structure of the modified recombinant hirudin is shown in figure 2; the solvent of the modified recombinant hirudin solution is water; the concentration of the modified recombinant hirudin solution is 1 mM; the reaction time is 360min, and the reaction temperature is 48 ℃;
(4) after the reaction, the sample is ultrasonically cleaned for 5min by using ethanol and deionized water respectively to obtain the anticoagulation titanium-based material.
Anti-protein adhesion experiments show that compared with an unmodified control group, the constructed material can inhibit the adhesion of 88.2% of human serum albumin and 65.3% of fibronectin;
an anti-platelet adhesion experiment shows that compared with an unmodified control group, the constructed material can inhibit the adhesion of platelets by 77.0 percent;
the hemolysis experiment result shows that compared with a positive control group, the hemolysis rate of the constructed material is 0.2%;
the cell compatibility experiment result of the vascular endothelial cells shows that the cell activity of the surface of the constructed material is improved by 8.2 percent compared with that of an unmodified control group within 24 hours.
Example 5
The preparation method of the anticoagulant material comprises the following steps:
(1) carrying out hydrofluoric acid treatment on the medical pure titanium material, wherein the concentration of hydrofluoric acid is 6%, and the treatment time is 2 min;
(2) carrying out plasma treatment on the sample subjected to acid treatment, wherein the atmosphere of the plasma treatment is oxygen, and the treatment time is 6 min;
(3) immersing the sample treated in the steps (1) and (2) into a modified recombinant hirudin solution for reaction, and carrying out self-assembly reaction; wherein the structure of the modified recombinant hirudin is shown in figure 3; the solvent of the modified recombinant hirudin solution is a 60% ethanol water solution; the concentration of the modified recombinant hirudin solution is 4 mM; the reaction time is 180min, and the reaction temperature is 37 ℃;
(4) after the reaction, the sample is ultrasonically cleaned for 5min by using ethanol and deionized water respectively to obtain the anticoagulation titanium-based material.
Anti-protein adhesion experiments showed that the constructed material was able to inhibit 66.7% human serum albumin and 52.5% fibronectin adhesion compared to the unmodified control;
an anti-platelet adhesion experiment shows that compared with an unmodified control group, the constructed material can inhibit 30.8% of platelet adhesion;
the hemolysis experiment result shows that compared with a positive control group, the hemolysis rate of the constructed material is 0.6%;
the cell compatibility experiment result of the vascular endothelial cells shows that the cell activity of the surface of the constructed material is improved by 6.3 percent compared with that of an unmodified control group within 24 hours.
Example 6
The preparation method of the anticoagulant material comprises the following steps:
(1) carrying out hydrofluoric acid treatment on the medical pure titanium material, wherein the concentration of hydrofluoric acid is 5%, and the treatment time is 2.5 min;
(2) carrying out plasma treatment on the sample subjected to acid treatment, wherein the atmosphere of the plasma treatment is air, and the treatment time is 6 min;
(3) immersing the sample treated in the steps (1) and (2) into a modified recombinant hirudin solution for reaction, and carrying out self-assembly reaction; wherein the structure of the modified recombinant hirudin is shown in figure 4; the solvent of the modified recombinant hirudin solution is a 50% ethanol water solution; the concentration of the modified recombinant hirudin solution is 0.3 mM; the reaction time is 300min, and the reaction temperature is 50 ℃;
(4) after the reaction, the sample is ultrasonically cleaned for 5min by using ethanol and deionized water respectively to obtain the anticoagulation titanium-based material.
Anti-protein adhesion experiments showed that the constructed material was able to inhibit the adhesion of 46.3% human serum albumin and 44.6% fibronectin compared to the unmodified control;
an anti-platelet adhesion experiment shows that compared with an unmodified control group, the constructed material can inhibit 37.3% of platelet adhesion;
the hemolysis experiment result shows that compared with a positive control group, the hemolysis rate of the constructed material is 0.5%;
the cell compatibility experiment result of the vascular endothelial cells shows that the cell activity of the surface of the constructed material is improved by 3.3 percent compared with that of an unmodified control group within 24 hours.
Example 7
The preparation method of the anticoagulant material comprises the following steps:
(1) carrying out hydrofluoric acid treatment on the medical pure titanium material, wherein the concentration of hydrofluoric acid is 5%, and the treatment time is 3 min;
(2) carrying out plasma treatment on the sample subjected to acid treatment, wherein the atmosphere of the plasma treatment is oxygen, and the treatment time is 3 min;
(3) immersing the sample treated in the steps (1) and (2) into a modified recombinant hirudin solution for reaction, and carrying out self-assembly reaction; wherein the structure of the modified recombinant hirudin is shown in figure 2; the solvent of the modified recombinant hirudin solution is a 50% ethanol water solution; the concentration of the modified recombinant hirudin solution is 8 mM; the reaction time is 360min, and the reaction temperature is 50 ℃;
(4) after the reaction, the sample is ultrasonically cleaned for 5min by using ethanol and deionized water respectively to obtain the anticoagulation titanium-based material.
Anti-protein adhesion experiments show that compared with an unmodified control group, the constructed material can inhibit the adhesion of 78.2% of human serum albumin and 77.1% of fibronectin;
an anti-platelet adhesion experiment shows that compared with an unmodified control group, the constructed material can inhibit 60.6% of platelet adhesion;
the hemolysis experiment result shows that compared with the positive control group, the hemolysis rate of the constructed material is 0.7%;
the cell compatibility experiment result of the vascular endothelial cells shows that the cell activity of the surface of the constructed material is improved by 8.0 percent compared with that of an unmodified control group within 24 hours.
Example 8
A preparation method of an anticoagulation titanium-based material comprises the following steps:
(1) carrying out hydrofluoric acid treatment on the medical pure titanium material, wherein the concentration of hydrofluoric acid is 9%, and the treatment time is 3 min;
(2) carrying out plasma treatment on the sample subjected to acid treatment, wherein the atmosphere of the plasma treatment is oxygen, and the treatment time is 10 min;
(3) immersing the sample treated in the steps (1) and (2) into a modified recombinant hirudin solution for reaction, and carrying out self-assembly reaction; wherein the structure of the modified recombinant hirudin is shown in figure 3; the solvent of the modified recombinant hirudin solution is 75% ethanol water solution; the concentration of the modified recombinant hirudin solution is 10 mM; the reaction time is 720min, and the reaction temperature is 50 ℃;
(4) after the reaction, the sample is ultrasonically cleaned for 5min by using ethanol and deionized water respectively to obtain the anticoagulation titanium-based material.
Anti-protein adhesion experiments show that compared with an unmodified control group, the constructed material can inhibit the adhesion of 82.1% of human serum albumin and 88.5% of fibronectin;
an anti-platelet adhesion experiment shows that compared with an unmodified control group, the constructed material can inhibit 90.3% of platelet adhesion;
the hemolysis experiment result shows that compared with the positive control group, the hemolysis rate of the constructed material is 0.7%;
the cell compatibility experiment result of the vascular endothelial cells shows that the cell activity of the surface of the constructed material is improved by 11.2 percent compared with that of an unmodified control group within 24 hours.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
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<120> anticoagulation titanium-based material and construction method thereof
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Claims (10)
1. A recombinant hirudin polypeptide comprising at least one of the following (i) to (IV):
(I) comprises at least one peptide fragment of VYTDCTESGLCCEGSHVCGQGNKCILGSDGEKNOCCETTCGTPKPQSHNDGDFEEIPEEYLO;
(ii) comprises at least one SKHKGGKHKGGKHKGKSSGK peptide stretch;
(iii) comprises at least one SKHKGGKHKGGKHKG peptide linked to a KSSGK peptide;
(IV) comprises at least one SKHKGGKHKGGKHKG peptide stretch linked to a tyrosine residue; at least one of the tyrosine residues is linked to a KSSGK peptide fragment.
2. The recombinant hirudin polypeptide of claim 1, which has the hirudin sequence of formula (I), and the polypeptide structure of any one of formulae (II) to (IV):
3. use of a polypeptide according to claim 1 or 2 for the preparation of an anticoagulant.
4. An anticoagulant material comprising a substrate material coated with a recombinant hirudin polypeptide of claim 1 or 2, wherein the recombinant hirudin polypeptide preferably comprises an amino acid group specifically binding to the titanium hydroxyl group.
5. The anticoagulant material of claim 4, wherein the recombinant hirudin polypeptide is attached to the surface of the base material via an amino-terminal sequence.
6. The anticoagulant material of claim 4 or 5 wherein the base material is pure titanium.
7. The method of preparing an anticoagulant material of any one of claims 4 to 6, comprising the steps of:
s1, carrying out acid treatment on a medical pure titanium material;
s2, carrying out plasma treatment on the acid-treated pure titanium material;
s3, immersing the pure titanium material treated in the step S2 into a solution containing the recombinant hirudin polypeptide to perform self-assembly reaction;
and S4, cleaning to obtain the anticoagulant material.
8. The method according to claim 7, wherein the acid treatment in step S1 is hydrofluoric acid treatment, the concentration of hydrofluoric acid is preferably 5% to 10%, and the treatment time is preferably 1 to 3 minutes;
the atmosphere of the plasma treatment in the step S2 is preferably oxygen or air, and the treatment time is preferably 1-10 min.
9. The method according to claim 7, wherein the solvent of the solution containing the recombinant hirudin polypeptide in step S3 is a mixture of water and ethanol, wherein the volume ratio of ethanol is 0% to 100%;
the concentration of the solution containing the recombinant hirudin polypeptide is preferably 0.01-10 mM;
the reaction time of the assembly reaction is preferably 20-720 min, and the reaction temperature is preferably 25-60 ℃.
10. Use of the anticoagulant material of any one of claims 4 to 6 in the preparation of a cardiovascular stent.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114057866A (en) * | 2021-12-23 | 2022-02-18 | 贵州省畜牧兽医研究所 | Leech polypeptide with antibacterial and hemolytic effects and application thereof |
| CN116328047A (en) * | 2022-12-22 | 2023-06-27 | 上海琦识医疗科技有限公司 | Precise medical catheter |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0649856A1 (en) * | 1993-10-19 | 1995-04-26 | Ciba-Geigy Ag | Process for the production of correct folded protein |
| CN1428173A (en) * | 2001-12-25 | 2003-07-09 | 重庆富进生物医药有限公司 | Recombinant hirudin oral enteric soluble slowly-releasing preparation |
| CN106139259A (en) * | 2016-08-17 | 2016-11-23 | 林春梅 | One has drug slow release function blood vessel bracket coating and preparation method thereof |
-
2021
- 2021-08-16 CN CN202110939931.8A patent/CN113817048B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0649856A1 (en) * | 1993-10-19 | 1995-04-26 | Ciba-Geigy Ag | Process for the production of correct folded protein |
| CN1428173A (en) * | 2001-12-25 | 2003-07-09 | 重庆富进生物医药有限公司 | Recombinant hirudin oral enteric soluble slowly-releasing preparation |
| CN106139259A (en) * | 2016-08-17 | 2016-11-23 | 林春梅 | One has drug slow release function blood vessel bracket coating and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 盛燕辉等: "壳聚糖/肝素和壳聚糖/重组水蛭素共价包被镍钛合金片的 体外生物相容性", 《中国组织工程研究与临床康复》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114057866A (en) * | 2021-12-23 | 2022-02-18 | 贵州省畜牧兽医研究所 | Leech polypeptide with antibacterial and hemolytic effects and application thereof |
| CN114057866B (en) * | 2021-12-23 | 2024-03-26 | 贵州省畜牧兽医研究所 | Leech polypeptide with antibacterial and hemolysis effects and application thereof |
| CN116328047A (en) * | 2022-12-22 | 2023-06-27 | 上海琦识医疗科技有限公司 | Precise medical catheter |
| CN116328047B (en) * | 2022-12-22 | 2023-10-10 | 上海琦识医疗科技有限公司 | Precise medical catheter |
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