CN107137757A - A kind of RGD bacteriophages/fibroin compound hemostatic material and preparation method thereof - Google Patents
A kind of RGD bacteriophages/fibroin compound hemostatic material and preparation method thereof Download PDFInfo
- Publication number
- CN107137757A CN107137757A CN201710284205.0A CN201710284205A CN107137757A CN 107137757 A CN107137757 A CN 107137757A CN 201710284205 A CN201710284205 A CN 201710284205A CN 107137757 A CN107137757 A CN 107137757A
- Authority
- CN
- China
- Prior art keywords
- rgd
- fibroin
- bacteriophages
- hemostatic material
- silk
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/10—Polypeptides; Proteins
- A61L24/108—Specific proteins or polypeptides not covered by groups A61L24/102 - A61L24/106
-
- 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
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
- A61L24/0015—Medicaments; Biocides
-
- 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
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
- A61L24/0036—Porous materials, e.g. foams or sponges
-
- 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
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
- A61L24/0042—Materials resorbable by the body
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- 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
-
- 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
- A61L2300/412—Tissue-regenerating or healing or proliferative 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
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/418—Agents promoting blood coagulation, blood-clotting agents, embolising 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
- A61L2300/602—Type of release, e.g. controlled, sustained, slow
- A61L2300/604—Biodegradation
-
- 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
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/04—Materials for stopping bleeding
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/00051—Methods of production or purification of viral material
- C12N2795/00052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Animal Behavior & Ethology (AREA)
- Surgery (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Organic Chemistry (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Materials Engineering (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Dispersion Chemistry (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention belongs to field of biomedical materials, a kind of preparation method of RGD bacteriophages/fibroin compound rapid hemostatic material of controlled degradation relate to.The invention provides a kind of natural biologic material for the biological absorbable being made up of silk-fibroin as support section, with reference to RGD bacteriophages/fibroin hemostatic material of the controlled degradation of RGD bacteriophages, blood coagulation effect is promoted with significant.It is mainly used in the hemostasis of skin wound, internal organs, is a kind of preferable hemostatic material.The present invention has following prominent characteristics：(1) fibroin albumen after handling, can attract blood platelet and red blood cell negatively charged in blood, increase platelet adhesion reaction and promotion thrombus；(2) after a certain time, RGD bacteriophages/fibroin cellular hemostatic material can degrade, then be absorbed by organisms so that wound location does not stay incrustation.(3) hemostatic capability of fibroin albumen is not depended on merely, by compound RGD bacteriophages, accelerates the anthemorrhagic speed and effect of hemostatic material.
Description
Technical field
The invention belongs to field of biomedical materials, the RGD bacteriophages/fibroin that relate to a kind of controlled degradation is compound quick
The preparation method of hemostatic material.
Background technology
It is fast that the wound hemostasis in emergency treatment, surgical procedure in provisional accident is required for suitable Hemostasis to carry out
Short stopping blood, and locally effectively quick-acting haemostatic powder is extremely important by patient, can effectively reduce casualties.The fast short stopping reported at present
Blood material category mainly has three classes：It is powdered (such as zeolite, collagen powder), tunica fibrosa (such as oxidized regenerated cellulose), many
Hole sponge material (such as gelfoam).But the mechanism of action and application method phase not to the utmost of hemostatic material that these raw materials are made
Together, hemostatic mainly by reinforcement intravascular coagulation factor or suppresses anticoagulant factor to promote blood coagulation, reach hemostasis purpose, stops blooding
Effect also has difference.Zeolite powder can be in wound site quick-acting haemostatic powder, but can be released after the moisture in absorbing blood big
The heat of amount, causes wound secondary insult and inflammatory reaction；Gelatin and collagen are poor to the adhesive force of haemocyte, and because collagen and
Gelatin is all the product extracted out of animal body, thus be accordingly used in the problem of there is immunogenicity among human body；Although studying above
Achievement can promote the development of hemostatic material and lift the performance of hemostatic material, but so far still well can be quick without one kind
The material of hemostasis.
With the continuous improvement required hemostatic material anthemorrhagic performance, develop anthemorrhagic speed faster, it is better only
Blood material is imperative.The platelet glycoprotein compound of activation is as by the adhesion egg such as physical efficiency identification and binding fiber proteinogen
RGD sequence (aspartic acid of one glycine of arginine one) common to white, causes platelet aggregation and thrombosis.
But in these related researchs, hemostatic factor or styptic are often chemical small molecule or are mechanical property
The porous material of shortcoming, is unsuitable for the hemostasis of large area wound.It is also unprecedented to enter RGD bacteriophages and silk fibroin water solution
Row is compound mixed, then prepare with excellent mechanical property, quick-acting haemostatic powder, high porosity hemostatic material, stop for human body wound
The report of blood.
Fibroin fiber is a kind of natural fiber albumen, and containing 18 kinds of amino acid to needed by human body, Juvenile Hormone is because of it
The mechanical property of uniqueness and good biocompatibility, controlled degradation, antibiotic property, stability, and non-immunogenicity, in life
Thing Material Field has a wide range of applications, and can such as be prepared into fibrous, membranaceous, cellular, gel, powdered a variety of shapes
Formula, the research applied to immobilised enzymes, antigen-antibody carrier, slow releasing carrier of medication and coagulant material etc..Fibroin albumen is in exploitation
There is bigger advantage in terms of rapid hemostatic material.Regenerated silk material is stronger to the adhesiveness of cell, epidermal cell, into fiber
Cell, mescenchymal stem cell and other adult cells can well grow on fibroin membrane, play its function.Fibroin divides greatly
Son constitutes the gel or fibrous network of high degree of hydration in cell peripheral, has support, adhesion to the specific cells group in blood, protects
The effect of shield.Meanwhile, silk fibroin protein solution can prepare 2 dimensions or 3-dimensional material by material preparation method, be hemostatic factor
Or styptic provides a suitable storage microenvironment, serves as the effect of " skeleton ".The general chemistry of bacteriophage construct trans
With the typical size of material science, there is superior dispersiveness in water.The displaying of its coat protein has substantial amounts of oxygen-content active base
Group, such as carbonyl, carboxyl, hydroxyl and epoxy radicals.Therefore, provided for the interaction between graphene oxide and fibroin albumen
Substantial amounts of binding site.Therefore the hemostatic support material based on fibroin albumen has good application prospect in technical field of biological material.
The content of the invention
For deficiency present in conventional fabrication techniques, the invention provides a kind of biological absorbable being made up of silk-fibroin
Natural biologic material as support section, with reference to RGD bacteriophages/fibroin hemostatic material of the controlled degradation of RGD bacteriophages, tool
There is significant promotion blood coagulation effect.It is mainly used in the hemostasis of skin wound, internal organs, is a kind of preferable hemostatic material.
A kind of RGD bacteriophages/fibroin compound hemostatic material, using natural macromolecular material fibroin material as carrier, with
RGD bacteriophages are core styptic, and the hemostatic support material of controlled degradation is made.
Wherein, fibroin final concentration in compound hemostatic material：0.001%~40%, RGD bacteriophage final concentration：102pfu/mL
~1020pfu/mL。
It is preferred that, the RGD bacteriophages include the hemostatic material of active component RGD peptide, and the RGD peptide sequence is:Smart ammonia
Acyl-glycyl-aspartic acid (Arg-Gly-Asp)；RGD peptide is obtained by display technique of bacteriophage, and method is as follows：1) phagocytosis
The structure of body display carrier；2) activation and amplification of TG1 cells；3) connect and convert TG1 cells；4) gene sequencing；5) phagocytosis
Body display and amplification.
In addition, the invention provides a kind of rapidity hemostatic gauze, being prepared by RGD bacteriophages/fibroin compound hemostatic material
Form.
Fibroin material hemostatic capability itself is limited, and the research of current fibroin albumen hemostatic material is the knot from material
Carried out in terms of structure, electric charge and interfacial property.Fibroin albumen is a kind of containing cationic amino acids such as lysine, arginine
Hmw protein, can produce certain electrostatic adsorption with electronegative blood platelet and red blood cell.And Application No.
A kind of patent of 201510953638.1 (RGD-M13 bacteriophages/oxidized regenerated cellulose oxidized regenerated celluloses), what it was used
It using oxidized regenerated cellulose is materials for support part that strategy, which is, and oxidized regenerated cellulose hemostasis principle is that acidic carboxypolymer can cause
Haemolysis so that erythrocyte dissolving, rupture, and discharge hemoglobin participation blood coagulation.Therefore silk-fibroin is used as blood coagulation material
Material does not destroy the activity of cell in blood, with the high characteristic of biocompatibility.The active group chemical characteristic of solidifying fibroin albumen
Difference, can carry out multiple reaction, while there is also ionic bond and hydrogen bond etc. with the antibody molecule of blood platelet, Surface of Erythrocytes
The combination of non-covalent bond.Due to the hydrophily of amino, the quantity of fibrinogen adsorption can be promoted, so that blood can be increased
Platelet adheres to and promotes thrombus, reaches haemostatic effect.Protein in silk fibroin material adsorption blood, excite blood platelet and
Clotting factor, so as to occur blood coagulation.Therefore, in terms of application study, a kind of carrier of styptic, a side can be used as with fibroin
The preservation of clotting factor can be improved and improved in face again, strengthen haemostatic effect.
High concentration fibroin of the present invention has good Combination with the RGD bacteriophages blending aqueous solution, is illustrated in figure 2 highly concentrated
Spend the atomic force microscopy figure of fibroin and the blending aqueous solution of RGD bacteriophages：Long fibre shape object is bacteriophage, and white particle is silk
Element, fibroin particle can be uniformly distributed in the surface (Fig. 2 B are Fig. 2A enlarged drawing) of bacteriophage fiber, obtained compound rest
The haemostatic effect of material is excellent, and with controllable degradation rate, is with a wide range of applications.
Present invention also offers a kind of preparation method of the RGD of controlled degradation bacteriophages/fibroin hemostatic material, technique in addition
Simply, handle convenient.
Technical scheme is as follows：
The preparation method of RGD bacteriophages/fibroin hemostatic material of a kind of controlled degradation, using following steps：
1) the fibrous fibroin obtained after degumming silkworm cocoons is sequentially passed through after dissolving, filtering, dialysis and centrifugation, is concentrated into matter
Measure the silk fibroin water solution that percentage is 1%~25%；
2) it is 10 by concentration7Pfu/mL~1015Pfu/mL phage solution ultrasound is disperseed, with 0.001%-40%
Silk fibroin protein solution is mixed, and is stirred, is obtained RGD bacteriophages/fibroin composite solution；
3) the RGD bacteriophages/fibroin for the three-dimensional honeycomb shape loose structure for obtaining different-shape using freeze-drying again is multiple
Close hemostatic material；
4) according to the degradation rate of required RGD bacteriophages/fibroin compound hemostatic material, in 30%-100% alcoholic solution
Middle processing 0.1-24h, the degradation rate of hemostatic material can control to degrade at 10min to 1 month.
The step 1) in fibroin raw material can also use silkworm silk gum, silk gland protein, wild silkworm silk-fibroin, spider silk fibroin
Or the silk-fibroin such as recombinant fibroin, but not limited to this.
The step 1) in fibroin mass percent be 0.001%~40% the aqueous solution, silk fibroin water solution matter
It is 0.10%~25% to measure percentage.
Step 2) described in the concentration of RGD- phage suspensions be：102Pfu/mL~1020Pfu/mL, preferentially using dense
Spend and be：107Pfu/mL~1015pfu/mL。
Step 2) described in RGD- bacteriophages be bacteriophage coat protein displaying have arginine-glycine-asparagus fern ammonia
The sequence of sour (RGD).
Wherein, the preparation method of RGD- bacteriophages, comprises the following steps：
1) structure of Vector for Phage Display, it is comprised the following steps that：
A. designing preceding primer and rear primer is used to preparing the corresponding base sequence of RGD Insert Fragment peptides：
Primer 1, introduces NcoI restriction enzyme sites：5′-ATCCATGGCGCGTGGCGACGATCCCGCAAAAGCG-3′；
Primer 2, introduces HindIII restriction enzyme sites：5′-GCAAGCTTTTATCAGCTTGCTTTCGAG-3′；
B. Primer 1 and Primer 1 are made into the single-stranded solution of oligonucleotide respectively, used by template of M13DNAPhusion archaeal dna polymerases expand whole coded sequence；
C. by expression plasmid digestion with restriction enzyme；
D. annealing oligonucleotide chain is connected into carrier with plasmid double digestion product in the case where T4DNA is connected enzyme effect, is obtained
Recombinant plasmid；
2) TG1 cells are arrived in the activation of TG1 cells and plasmid restructuring；
A. TG1 cells are seeded in LB culture mediums, 37 DEG C, 220r/min culture 4h activation；With step 1) in restructuring
Plasmid translation table reaches Host Strains TG1 cells, and 37 DEG C are continued to cultivate, and obtain competent cell；
B. picking single bacterium colony is used for amplifying cells, and plasmid extraction kit extracts plasmid order-checking to verify plasmid as the positive,
Prove that RGD gene orders are expressed in TG1 cells, obtain competence TG1 cells；
3) RGD Phage amplifications and purifying
By step 2) in competence TG1 cells 37 DEG C cultivate 2 hours after add helper phage, add IPTG
It is used as derivant, incubated overnight；High speed centrifugation collects supernatant and removes TG1 cells afterwards；PEG/NaCl is precipitated, precipitation is collected by centrifugation
RGD phage particles are obtained, the step 2 time is repeated, RGD bacteriophages dissolve scattered, obtain RGD bacteriophage water in deionized water
Solution, bacteriophage quantity is calculated by UV spectrum；
4) RGD bacteriophages are freeze-dried；
By step 3) in the RGD bacteriophage aqueous solution in freeze drier -40 DEG C be freeze-dried 12-24 hours, obtain
RGD bacteriophage powders.
Wherein, the polypeptide display is in the coat protein of p VIII of bacteriophage, or pIII, pVI, pIX or pVII protein loci
On.
The step 3) obtained RGD bacteriophage concentration of aqueous solution is：105Pfu/mL~1015pfu/mL。
The step 3) obtained RGD bacteriophage concentration of aqueous solution is：1011Pfu/mL~1013pfu/mL。
This patent gives the preparation technology and optimum condition of rapid hemostatic material., can be with according to different type of surgery
For the hemostasis of skin and the Different Organs surface of a wound, the surgical wound surface of such as tumor resection or the wound of bone tissue traumatic rupture
Mouthful, while hemostatic function is played, play a part of promoting injury tissue Regeneration and Repair, its haemostatic effect, which is better than, individually to be made
With fibroin material, thrombotest shows, the rapidity hemostatic gauze of preparation, high with quick-acting haemostatic powder, biocompatibility
Feature.By further clinical experimental study, it is expected to provide a kind of new absorbable hemostatic product.
Compared with prior art, the present invention has following prominent characteristics：
(1) fibroin albumen after handling, can attract blood platelet and red blood cell negatively charged in blood, and increase blood platelet glues
Echo promotion thrombus；
(2) after a certain time, RGD bacteriophages/fibroin cellular hemostatic material can degrade, then be absorbed by organisms, and make
Obtain wound location and do not stay incrustation.
(3) hemostatic capability of fibroin albumen is not depended on merely, by compound RGD bacteriophages, accelerates stopping for hemostatic material
Blood speed and effect.
Brief description of the drawings
Fig. 1 is the N- ends schematic diagram that RGD peptide is illustrated in the albumen of phage ghost P VIII.
Fig. 2 is the atomic force microscopy figure of high concentration fibroin and the blending aqueous solution of RGD bacteriophages.
Embodiment
Below by embodiment, the present invention is described in further detail, following examples be explanation of the invention and
The invention is not limited in following examples.
Bacteriophage styptic used herein is prepared by applicant, and specific method is as follows：
1) structure of Vector for Phage Display, it is comprised the following steps that：
A. designing preceding primer and rear primer is used to preparing the corresponding base sequence of RGD Insert Fragment peptides：
Primer 1：5 '-ATCCATGGCGCGTGGCGACGATCCCGCAAAAGCG-3 ' (introduce NcoI restriction enzyme sites)
Primer 2：5 '-GCAAGCTTTTATCAGCTTGCTTTCGAG-3 ' (introduce HindIII restriction enzyme sites)；
B. Primer 1 and Primer 1 are made into the single-stranded solution of oligonucleotide respectively, it is whole by template amplification of M13DNA
Individual coded sequence (is usedPhusion archaeal dna polymerases)；
C. by expression plasmid digestion with restriction enzyme；
D. annealing oligonucleotide chain is connected into carrier with plasmid double digestion product in the case where T4DNA is connected enzyme effect, is obtained
Recombinant plasmid.
2) TG1 cells are arrived in the activation of TG1 cells and plasmid restructuring；
A. TG1 cells are seeded in LB culture mediums, 37 DEG C, 220r/min culture 4h activation.With step 1) in restructuring
Plasmid translation table reaches Host Strains TG1 cells, and 37 DEG C are continued to cultivate, and obtain competent cell；
B. picking single bacterium colony is used for amplifying cells, and plasmid extraction kit extracts plasmid order-checking to verify plasmid as the positive,
Prove that RGD gene orders are expressed in TG1 cells, obtain competence TG1 cells.
3) RGD Phage amplifications and purifying
By step 2) in competence TG1 cells 37 DEG C cultivate 2 hours after add helper phage, add IPTG
It is used as derivant, incubated overnight.High speed centrifugation collects supernatant and removes TG1 cells afterwards.PEG/NaCl is precipitated, precipitation is collected by centrifugation
RGD phage particles are obtained, the step 2 time is repeated, RGD bacteriophages dissolve scattered, obtain RGD bacteriophage water in deionized water
Solution, bacteriophage quantity is calculated by UV spectrum.
4) RGD bacteriophages are freeze-dried；
By step 3) in the RGD bacteriophage aqueous solution in freeze drier -40 DEG C be freeze-dried 24 hours, obtain RGD
Bacteriophage powder (such as Fig. 1).
Embodiment 1
The preparation method of RGD bacteriophages/fibroin hemostatic material of controlled degradation
1) the fibrous fibroin obtained after degumming silkworm cocoons is sequentially passed through after dissolving, filtering, dialysis and centrifugation, is concentrated into matter
Measure the silk fibroin water solution that percentage is 100mg/mL；
2) it is 10 by concentration7Pfu/mL phage solution ultrasound is disperseed, and is mixed with silk fibroin protein solution, stirring is equal
It is even, obtain RGD bacteriophages/fibroin composite solution；
3) in temperature it is again vacuum freeze drying 24h at -20 DEG C, obtains three-dimensional porous RGD bacteriophages/fibroin compound only
Blood material；
4) 30% ethanol postincubation, enable to the degradation rate of three-dimensional porous RGD bacteriophages/fibroin hemostatic material
Control is in one day.
Embodiment 2
The preparation method of RGD bacteriophages/fibroin hemostatic material of controlled degradation, comprises the following steps：
1) the fibrous fibroin obtained after degumming silkworm cocoons is sequentially passed through after dissolving, filtering, dialysis and centrifugation, is concentrated into matter
Measure the silk fibroin water solution that percentage is 1mg/mL；
2) it is 10 by concentration14Pfu/mL phage solution ultrasound is disperseed, and is mixed with silk fibroin protein solution, stirring is equal
It is even, obtain RGD bacteriophages/fibroin composite solution；
3) in temperature it is again vacuum freeze drying 48h at -80 DEG C, obtains three-dimensional porous RGD bacteriophages/fibroin compound only
Blood material；
4) 80% ethanol postincubation, enable to three-dimensional porous RGD bacteriophages/fibroin hemostatic material degradation rate base
This does not occur degraded (dissolve-loss ratio is more than 80% after 1 month).
Embodiment 3
The preparation method of RGD bacteriophages/fibroin hemostatic material of controlled degradation, using following steps：
1) the fibrous fibroin obtained after degumming silkworm cocoons is sequentially passed through after dissolving, filtering, dialysis and centrifugation, is concentrated into matter
Measure the silk fibroin water solution that percentage is 0.1mg/mL；
2) it is 10 by concentration20Pfu/mL phage solution ultrasound is disperseed, and is mixed with silk fibroin protein solution, stirring is equal
It is even, obtain RGD bacteriophages/fibroin composite solution；
3) three-dimensional porous RGD bacteriophages/fibroin compound hemostatic material is obtained using freeze-drying again；
4) new fresh rabbit blood 2mL is taken, RGD bacteriophages/fibroin compound hemostatic material of preparation is put into new fresh rabbit blood, 3 points
Zhong Houxin fresh rabbit blood solidifies.
Thus, present invention process is simple, and the i.e. generation Supramolecular self assembly of contact is made between RGD bacteriophages and fibroin albumen
Hemostatic material；Hemostatic material made from the invention has good quick-acting haemostatic powder effect, and can regulate and control bacteriophage/fibroin and stop
The degradation speed of blood material, the shapeless problem for overcoming prior art to exist, is suitable for industrialized production；The RGD finally given
Bacteriophage/fibroin hemostatic material is widely used, available for operation after hemostatic material, wound dressing use, contribute to the surface of a wound
Heal and non-stimulated and environment friendly and pollution-free, with prominent significant technique effect.
Finally, in addition it is also necessary to it is noted that listed above is only specific embodiment of the invention.Obviously, the present invention is not limited
In above example, there can also be many deformations.One of ordinary skill in the art can directly lead from present disclosure
All deformations for going out or associating, are considered as protection scope of the present invention.
Claims (9)
1. a kind of RGD bacteriophages/fibroin compound hemostatic material, be using natural macromolecular material fibroin material as carrier, with
RGD bacteriophages are core styptic, the hemostatic support material for the controlled degradation being made.
2. RGD bacteriophages/fibroin compound hemostatic material according to claim 1, it is characterised in that：Silk in compound hemostatic material
Plain final concentration：0.001%~40%, RGD bacteriophage final concentration：102Pfu/mL~1020pfu/mL。
3. RGD bacteriophages/fibroin compound hemostatic material according to claim 1, it is characterised in that：The RGD bacteriophages are
The hemostatic material of active component RGD peptide is included, the RGD peptide sequence is:Arginyl-glycyl-aspartic acid (Arg-Gly-
Asp)；RGD peptide is obtained by display technique of bacteriophage, and method is as follows：1) structure of Vector for Phage Display；2) TG1 cells
Activation and amplification；3) connect and convert TG1 cells；4) gene sequencing；5) phage display and amplification.
4. a kind of rapidity hemostatic gauze, as described in claim 1 or 2 or 3 prepared by RGD bacteriophages/fibroin compound hemostatic material
Form.
5. the preparation method of RGD bacteriophages/fibroin compound hemostatic material described in claim 1 or 2 or 3, it is characterised in that：Using
Following steps：
1) fibrous fibroin is sequentially passed through after dissolving, filtering, dialysis and centrifugation, be concentrated into mass percent for 0.001%~
40% silk fibroin water solution；
2) it is 10 by concentration2Pfu/mL~1020Pfu/mL RGD phage solutions ultrasound is disperseed, with 0.001%-40%
Silk fibroin protein solution is mixed, and is stirred, is obtained RGD bacteriophages/fibroin composite solution；Described RGD bacteriophages are in phagocytosis
Body case protein display has arginine-glycine-aspartic acid (RGD) sequence；
3) by step 3) RGD bacteriophages/fibroin composite solution obtains the different-shapes of different patterns using freeze-drying
RGD bacteriophages/fibroin compound hemostatic material of three-dimensional honeycomb shape loose structure；
4) by step 3) in RGD bacteriophages/fibroin compound hemostatic material needed for RGD bacteriophages/fibroin compound hemostatic material
The degradation rate of material, 0.1-24h is handled in 30%-100% alcoholic solution.
6. preparation method according to claim 5, it is characterised in that：The step 1) in fibrous fibroin raw material be silkworm
Cocoon degumming and obtain, or silkworm silk gum, silk gland protein, wild silkworm silk-fibroin, spider silk fibroin or recombinant fibroin.
7. preparation method according to claim 5, it is characterised in that：The step 1) in silk fibroin water solution quality
Percentage is 0.10%~25%.
8. preparation method according to claim 5, it is characterised in that：Step 2) described in RGD phage solutions concentration
For：107pfu/mL~1014pfu/mL.
9. preparation method according to claim 5, it is characterised in that：The step 2) described in RGD- bacteriophages be
Bacteriophage coat protein shows the sequence for having arginine-glycine-aspartic acid (RGD).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710284205.0A CN107137757A (en) | 2017-04-26 | 2017-04-26 | A kind of RGD bacteriophages/fibroin compound hemostatic material and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710284205.0A CN107137757A (en) | 2017-04-26 | 2017-04-26 | A kind of RGD bacteriophages/fibroin compound hemostatic material and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107137757A true CN107137757A (en) | 2017-09-08 |
Family
ID=59773991
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710284205.0A Pending CN107137757A (en) | 2017-04-26 | 2017-04-26 | A kind of RGD bacteriophages/fibroin compound hemostatic material and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107137757A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109157672A (en) * | 2018-08-16 | 2019-01-08 | 浙江大学 | A kind of preparation method of fibroin-small peptide blood coagulation microballoon |
| CN110305202A (en) * | 2019-07-03 | 2019-10-08 | 浙江大学 | A method of improving fibroin albumen biomineralisation capabilities |
| CN110448719A (en) * | 2019-06-28 | 2019-11-15 | 浙江大学 | A kind of fibroin-polypeptide electrospinning film and preparation method thereof promoting blood coagulation |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1132515A (en) * | 1993-09-30 | 1996-10-02 | 新日本制铁株式会社 | Novel peptide, active as inhibitors of platelet aggregation |
| CN102743796A (en) * | 2011-04-18 | 2012-10-24 | 北京汇亨创管理咨询有限公司 | Silk fibroin porous support made from polyvinyl alcohol, and preparation method and application thereof |
| CN103418022A (en) * | 2013-07-15 | 2013-12-04 | 江苏海泽医疗科技发展有限公司 | Composite multifunctional biological hemostatic material and preparation method thereof |
| CN105435295A (en) * | 2015-12-15 | 2016-03-30 | 哈尔滨工业大学 | Preparation method of RGD-M13 bacteriophage/oxidized regenerated cellulose composite hemostatic material |
| CN105477675A (en) * | 2015-12-15 | 2016-04-13 | 哈尔滨工业大学 | Preparation method of RGD-M13 phage/polylysine/oxidized regenerated cellulose composite haemostatic material |
| CN105521520A (en) * | 2015-12-10 | 2016-04-27 | 苏州大学 | Preparation method of silkworm fibroin hemostatic material |
-
2017
- 2017-04-26 CN CN201710284205.0A patent/CN107137757A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1132515A (en) * | 1993-09-30 | 1996-10-02 | 新日本制铁株式会社 | Novel peptide, active as inhibitors of platelet aggregation |
| CN102743796A (en) * | 2011-04-18 | 2012-10-24 | 北京汇亨创管理咨询有限公司 | Silk fibroin porous support made from polyvinyl alcohol, and preparation method and application thereof |
| CN103418022A (en) * | 2013-07-15 | 2013-12-04 | 江苏海泽医疗科技发展有限公司 | Composite multifunctional biological hemostatic material and preparation method thereof |
| CN105521520A (en) * | 2015-12-10 | 2016-04-27 | 苏州大学 | Preparation method of silkworm fibroin hemostatic material |
| CN105435295A (en) * | 2015-12-15 | 2016-03-30 | 哈尔滨工业大学 | Preparation method of RGD-M13 bacteriophage/oxidized regenerated cellulose composite hemostatic material |
| CN105477675A (en) * | 2015-12-15 | 2016-04-13 | 哈尔滨工业大学 | Preparation method of RGD-M13 phage/polylysine/oxidized regenerated cellulose composite haemostatic material |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109157672A (en) * | 2018-08-16 | 2019-01-08 | 浙江大学 | A kind of preparation method of fibroin-small peptide blood coagulation microballoon |
| CN110448719A (en) * | 2019-06-28 | 2019-11-15 | 浙江大学 | A kind of fibroin-polypeptide electrospinning film and preparation method thereof promoting blood coagulation |
| WO2020259672A1 (en) * | 2019-06-28 | 2020-12-30 | 浙江大学 | Fibroin-polypeptide electrospun film for promoting blood coagulation and preparation method therefor |
| US12121626B2 (en) | 2019-06-28 | 2024-10-22 | Zhejiang University | Blood coagulation-promoting silk fibroin-polypeptide electrospun membrane and preparation method thereof |
| CN110305202A (en) * | 2019-07-03 | 2019-10-08 | 浙江大学 | A method of improving fibroin albumen biomineralisation capabilities |
| CN110305202B (en) * | 2019-07-03 | 2021-06-08 | 浙江大学 | Method for improving biomineralization capability of silk fibroin |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104857569A (en) | Preparation method of fibroin and graphene oxide composite bracket material | |
| CN105688274B (en) | A kind of preparation process of polycaprolactone/gelatin electrospinning compound rest | |
| WO2013105665A1 (en) | Collagen structure, and method for producing collagen structure | |
| CN102143765B (en) | Acellular matrix glue | |
| CN103463668A (en) | Preparation method of porous dressing of silk fibroin-calcium alginate biological wound | |
| JP2002511284A (en) | Creating a three-dimensional organization | |
| CN107137757A (en) | A kind of RGD bacteriophages/fibroin compound hemostatic material and preparation method thereof | |
| CN103041445A (en) | Method for preparing molecular imprinting porous gel membrane for tissue engineering | |
| CN110548171B (en) | Gelatin-based bone tissue adhesive, and preparation method and application thereof | |
| CN102743796A (en) | Silk fibroin porous support made from polyvinyl alcohol, and preparation method and application thereof | |
| CN106880871B (en) | Collagen dermal material for promoting endometrial repair and preparation method thereof | |
| Yuan et al. | Fabrication of self-assembling peptide nanofiber hydrogels for myocardial repair | |
| CN1197631C (en) | Construction method for skin tissue engineering rack containing epidermal growth factor | |
| CN107823699B (en) | Hemostatic anti-adhesion membrane and preparation method thereof | |
| US12121626B2 (en) | Blood coagulation-promoting silk fibroin-polypeptide electrospun membrane and preparation method thereof | |
| CN106421884A (en) | Method for preparing hemostatic sponge by two-step freezing method | |
| Dong et al. | Electrospun nanofibrous membranes of recombinant human collagen type III promote cutaneous wound healing | |
| Luo et al. | Development and evaluation of tilapia skin-derived gelatin, collagen, and acellular dermal matrix for potential use as hemostatic sponges | |
| CN114225113B (en) | Degradable artificial dura mater with double-layer structure and preparation method thereof | |
| CN102772822A (en) | Application of collagen matrix as tissue engineering scaffold | |
| CN106913903A (en) | A kind of preparation method for loading Bioglass skin regeneration material | |
| Cui et al. | Polydopamine-coated polycaprolactone electrospun nanofiber membrane loaded with thrombin for wound hemostasis | |
| US20150037432A1 (en) | Self-assembly of collagen fibers from dermis, fascia and tendon for tissue augmentation and coverage of wounds and burns | |
| CN109513037B (en) | Mesoporous bioglass-loaded small intestine submucosa wound dressing | |
| WO2009122710A1 (en) | Medical composition |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20170908 |