CN110041557B - Functional silk fibroin porous material or functional silk fibroin membrane and preparation method thereof - Google Patents

Functional silk fibroin porous material or functional silk fibroin membrane and preparation method thereof Download PDF

Info

Publication number
CN110041557B
CN110041557B CN201910304083.6A CN201910304083A CN110041557B CN 110041557 B CN110041557 B CN 110041557B CN 201910304083 A CN201910304083 A CN 201910304083A CN 110041557 B CN110041557 B CN 110041557B
Authority
CN
China
Prior art keywords
fibroin
silk fibroin
functional
aqueous solution
porous material
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.)
Active
Application number
CN201910304083.6A
Other languages
Chinese (zh)
Other versions
CN110041557A (en
Inventor
王建南
宋广州
李荷雷
裔洪根
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suzhou University
Original Assignee
Suzhou University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Suzhou University filed Critical Suzhou University
Priority to CN201910304083.6A priority Critical patent/CN110041557B/en
Publication of CN110041557A publication Critical patent/CN110041557A/en
Application granted granted Critical
Publication of CN110041557B publication Critical patent/CN110041557B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
    • C08H1/00—Macromolecular products derived from proteins
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J5/00—Manufacture of articles or shaped materials containing macromolecular substances
    • C08J5/18—Manufacture of films or sheets
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
    • C08J7/12—Chemical modification
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
    • C08J9/28—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a liquid phase from a macromolecular composition or article, e.g. drying of coagulum
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
    • C08J9/36—After-treatment
    • C08J9/40—Impregnation
    • 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
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2201/00—Foams characterised by the foaming process
    • C08J2201/04—Foams characterised by the foaming process characterised by the elimination of a liquid or solid component, e.g. precipitation, leaching out, evaporation
    • C08J2201/048—Elimination of a frozen liquid phase
    • C08J2201/0484—Elimination of a frozen liquid phase the liquid phase being aqueous
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2389/00—Characterised by the use of proteins; Derivatives thereof

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Polymers & Plastics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Epidemiology (AREA)
  • Materials Engineering (AREA)
  • Dermatology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Manufacturing & Machinery (AREA)
  • Vascular Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Dispersion Chemistry (AREA)
  • Biochemistry (AREA)
  • Cosmetics (AREA)
  • Manufacture Of Porous Articles, And Recovery And Treatment Of Waste Products (AREA)
  • Treatments For Attaching Organic Compounds To Fibrous Goods (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses a functional silk fibroin porous material or a functional silk fibroin membrane and a preparation method thereof. The product prepared by the method is loaded with an active peptide which has the functions of regulating the competitive growth of vascular cells and activating vasodilatation; the adhesion and proliferation of endothelial cells are remarkably promoted, and the proliferation of smooth muscle cells is inhibited to a certain extent; in particular, the functional group or structural domain of the active peptide which plays the active role is protected by adopting the electrostatic binding force principle from influencing the activity of the active peptide due to chemical reaction. And the prepared functional fibroin porous material or membrane belongs to a non-hemolytic material, and the hemolytic rate is less than 0.4%.

Description

Functional silk fibroin porous material or functional silk fibroin membrane and preparation method thereof
Technical Field
The invention relates to the technical field of preparation of a silk fibroin material loaded with bioactive factors, in particular to a functional silk fibroin porous material or a functional silk fibroin membrane and a preparation method thereof.
Background
Silkworm silk is natural animal protein synthesized and secreted by silkworm, has wide source, and the silk fibroin has good biocompatibility and consists of 20 amino acids absorbable by human body, and the final degradation product is amino acid or small peptide which is easily absorbed or phagocytized by cells and can not cause obvious immune reaction. A great deal of research shows that the silk fibroin material can support the growth of various cells and contains a great amount of-COOH and NH2Functional groups such as-OH and the like provide chemical structure basis of various biological activity modification, and are increasingly researched and applied in the field of tissue regeneration medicine.
The invention aims to develop a silk fibroin functional material loaded with calcitonin gene-related peptide (CGRP) and applied to regeneration and repair of vascular tissues. CGRP is an active peptide consisting of 37 amino acids and widely distributed in the central and peripheral nervous systems, and is an important transmitter for transmitting information in the central and peripheral nervous systems. The nervous system is a system that is distributed throughout the body to regulate various physiological functions and behavioral activities of the body. In particular, CGRP is the most potent vasoactive peptide found in vivo and has important roles in regulating blood pressure, protecting the heart and preventing coronary atherosclerosis. Almost all blood vessels have nerve fibers with CGRP distributed therein. CGRP active peptide has the functions of stimulating vasodilatation, promoting the proliferation of vascular endothelial cells and transferring to damaged vascular walls. The CGRP active peptide is used as a protective component, and also has the functions of inhibiting the proliferation and migration of vascular smooth muscle cells, participating in the repair of vascular injury and the like. Therefore, the CGRP active peptide loaded into the vascular tissue engineering scaffold has great application prospect in regulating and controlling the regeneration or functional repair of defective blood vessels.
Disclosure of Invention
The invention aims to provide a functional silk fibroin porous material or a functional silk fibroin film and a preparation method thereof, which are developed aiming at easy thrombosis formation of vascular tissue engineering and low early-stage diastolic activity of vascular tissue regeneration so as to promote tissue regeneration and function recovery of diseased and defected blood vessels.
The invention has a technical scheme that:
provides a functional fibroin porous material or a functional fibroin membrane loaded with CGRP.
The other technical scheme of the invention is as follows:
a preparation method of a functional fibroin porous material or a functional fibroin membrane is provided, which comprises the following steps:
(1) preparing degummed silkworm fibroin fibers: placing silkworm silk or cocoon shells into a sodium carbonate aqueous solution, heating, cleaning, loosening, and drying to obtain degummed silkworm fibroin fibers;
(2) preparing a silkworm fibroin dissolving solution: completely dissolving the degummed bombyx mori silk fibroin fibers in a lithium bromide aqueous solution to obtain a silk fibroin dissolving solution;
(3) preparing a purified silkworm silk fibroin aqueous solution: filling the fibroin dissolving solution into a dialysis bag, then placing the dialysis bag into a container filled with deionized water, replacing the liquid in the container with new deionized water or pure water every 2 hours, continuously dialyzing for 3 days, and concentrating to obtain a purified silkworm fibroin aqueous solution;
(4) preparing a modified fibroin porous material or a modified fibroin membrane: adding adipic acid into the purified bombyx mori silk fibroin aqueous solution, then adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide, then adding 2-morpholine ethanesulfonic acid, stirring in an ice bath for 10-30 minutes, freeze-drying or drying, placing the freeze-dried or dried material into a container containing deionized water, soaking for 24-48 hours, replacing the deionized water in the container every 2-4 hours, taking out, and freeze-drying or air-drying again to obtain a modified silk fibroin porous material or a modified fibroin membrane;
(5) preparing a functional silk fibroin porous material and a functional silk fibroin film: and soaking the modified silk fibroin porous material in a CGRP aqueous solution with positive charges, or adding the CGRP aqueous solution with positive charges on the modified silk fibroin membrane, standing, collecting the solution, and finally drying the material at room temperature (<25 ℃) by air to obtain the functional silk fibroin porous material or the functional silk fibroin membrane.
Further, the step (1) of putting the silkworm silk or the cocoon shell into the sodium carbonate aqueous solution means that the silkworm silk or the cocoon shell is put into the sodium carbonate aqueous solution with the mass concentration of 0.2-0.8% according to the bath ratio of 1g to 50 mL; the heating treatment is carried out for 2-3 times at 98-100 ℃, each time for 30 minutes, and the drying is carried out in a 60 ℃ oven.
Further, the step (2) of completely dissolving the degummed bombyx mori silk fibroin fibers in the lithium bromide aqueous solution means that the degummed bombyx mori silk fibroin fibers are weighed, dissolved in 9.3M lithium bromide aqueous solution according to a bath ratio of 1g:10mL, and treated at the temperature of 65 ℃ until the silk fibroin fibers are completely dissolved.
Further, the dialysis bag in the step (3) is a semipermeable membrane, the molecular weight cutoff is 10-14 kDa, and the concentration is specifically performed by adopting a rotary evaporator, so that the mass fraction of the dialyzed bombyx mori silk fibroin aqueous solution is 5-15%.
Further, the mass ratio of the silkworm silk fibroin purified in the step (4) to the adipic acid is 100: 1-20, the addition amount of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is 30-50% of the mass of the purified silkworm silk fibroin, the addition amount of the N-hydroxysuccinimide is 15-25% of the mass of the purified silkworm silk fibroin, and the final concentration of the 2-morpholine ethanesulfonic acid is 0.05M.
Further, the freeze-drying in the step (4) is specifically as follows: pre-freezing for 2-24 hours at the temperature of-80 ℃ to-20 ℃, and then putting into a freeze dryer for freeze-drying.
Further, the drying in the step (4) is specifically to pour the mixed solution into a flat polystyrene plate and dry the mixed solution at 40-60 ℃.
Further, in the step (5), when the modified fibroin porous material is immersed in an aqueous solution of CGRP with positive charges, the final concentration of the aqueous solution of CGRP is 10-1000 nM; when the CGRP aqueous solution with positive charges is added to the modified silk fibroin film, the concentration of the CGRP aqueous solution is 50-1000 ng/cm2。
Further, the standing in the step (5) is carried out for 6-10 hours at the temperature of 4 ℃.
The invention provides a functional fibroin porous material or a functional fibroin membraneThe prepared product carries an active peptide which has the functions of regulating the competitive growth of vascular cells and activating vasodilatation; the adhesion and proliferation of endothelial cells are remarkably promoted, and the proliferation of smooth muscle cells is inhibited to a certain extent; especially, the electrostatic binding force principle is adopted to protect the functional groups (-COOH, -NH) of the active peptide which plays the active role2OH) or domains do not affect their activity by being chemically reacted. And the prepared functional fibroin porous material or membrane belongs to a non-hemolytic material (hemolytic rate)<0.4 percent) of the fibroin porous material or membrane, the functional fibroin porous material or membrane obtained by the invention has the functions of rapid endothelialization and hyperplasia inhibition, the rapid endothelialization is the fundamental factor for inhibiting thrombosis, and the fibroin porous material or membrane has wide application prospect in vascular tissue engineering.
Detailed Description
The invention provides a preparation method of a functional fibroin porous material or a functional fibroin membrane, which comprises the following steps:
step one, preparing degummed silkworm fibroin fibers:
the silkworm silk or cocoon shells are put into 0.2% sodium carbonate aqueous solution according to the bath ratio of 1g:50mL, treated for 3 times at the temperature of 98-100 ℃, treated for 30 minutes each time, cleaned, pulled loose and dried in a 60 ℃ oven, and the degummed silkworm fibroin fibers are obtained.
Step two, preparing a bombyx mori fibroin dissolving solution:
weighing the degummed bombyx mori silk fibroin fibers, dissolving the degummed bombyx mori silk fibroin fibers in 9.3M lithium bromide aqueous solution according to a bath ratio of 1g:10mL, and treating at the temperature of 65 ℃ until the silk fibroin fibers are completely dissolved to obtain a silk fibroin dissolving solution.
Step three, preparing the purified silkworm fibroin aqueous solution:
and (2) filling the fibroin dissolving solution into a dialysis bag, wherein the dialysis bag is a semipermeable membrane, the molecular weight cutoff is 10-14 kDa, then placing the dialysis bag into a container filled with deionized water, replacing the liquid in the container with new deionized water or pure water every 2 hours, continuously dialyzing for 3 days, and concentrating by using a rotary evaporator to obtain the purified bombyx mori silk fibroin aqueous solution with the mass fraction of 5-15%.
Step four, preparing the modified fibroin porous material or the modified fibroin film:
the method comprises the following steps: adding adipic acid into the purified silkworm fibroin aqueous solution, wherein the mass ratio of the purified silkworm fibroin to the adipic acid is 100: 1-20, then adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide, the addition amount of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is 30-50% of the mass of the purified silkworm fibroin, the addition amount of the N-hydroxysuccinimide is 15-25% of the mass of the purified silkworm fibroin, then adding 2-morpholine ethanesulfonic acid, the final concentration of the 2-morpholine ethanesulfonic acid is 0.05M, stirring for 10-30 minutes in an ice bath, pre-freezing for 2-24 hours at the temperature of-80-20 ℃, and freeze-drying in a freeze dryer, taking out the silk fibroin porous material, placing the silk fibroin porous material in a container filled with deionized water, soaking for 24-48 hours, replacing the deionized water in the container every 2-4 hours, and finally freeze-drying again to obtain the modified silk fibroin porous material.
The second method comprises the following steps: adding adipic acid into the purified silkworm fibroin aqueous solution, wherein the mass ratio of the purified silkworm fibroin to the adipic acid is 100: 1-20, then adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide, the adding amount of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is 30-50% of the mass of the purified silkworm fibroin, the adding amount of the N-hydroxysuccinimide is 15-25% of the mass of the purified silkworm fibroin, then adding 2-morpholine ethanesulfonic acid, the final concentration of the 2-morpholine ethanesulfonic acid is 0.05M, stirring for 10-30 minutes in an ice bath, pouring the mixed solution into a flat polystyrene flat dish, drying at 40-60 ℃, taking out, placing into a container containing deionized water, and soaking for 24-48 hours, and replacing the deionized water in the container every 2-4 hours, and finally air-drying to obtain the modified silk fibroin film.
And step five, preparing the functional fibroin porous material or the functional fibroin membrane.
The method comprises the following steps: the modified silk fibroin porous material is soaked in an aqueous solution (10-1000 nM) of CGRP (calcitonin gene-related peptide) with positive charges, placed at 4 ℃ for 6-10 hours to enable the CGRP to be adsorbed and deposited on the silk fibroin material, finally taken out and squeezed to be dry, and air-dried at room temperature (25 ℃), and the functional silk fibroin porous material is obtained.
The second method comprises the following steps: putting the modified silk fibroin film back to a plate for paving, and adding the silk fibroin film with the concentration of 50-1000 ng/cm2The aqueous solution of CGRP (1) is left at 4 ℃ for 6 to 10 hours and then taken out at room temperature<Air drying at 25 deg.C to obtain functional silk fibroin film.
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, embodiments accompanying the present invention are further described below. The invention is not limited to the embodiments listed but also comprises any other known variations within the scope of the invention as claimed.
First, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1
The embodiment shows a preparation method of a functional fibroin porous material, which comprises the following steps:
1. the method comprises the steps of putting raw silkworms or cocoon shells of silkworms into a sodium carbonate aqueous solution with the concentration of 0.2% according to the bath ratio of 1:50(g/mL), processing for three times at the temperature of 98-100 ℃ for 30 minutes each time, then fully cleaning the silks with deionized water, loosening the silks, and drying the silks in an oven at the temperature of 60 ℃ to obtain the degummed silk fibroin fibers of the silkworms.
2. Weighing degummed fibroin, dissolving in 9.3M lithium bromide water solution according to a bath ratio of 1:10(g/mL), and treating at 65 deg.C until fibroin fiber is completely dissolved to obtain domestic silkworm fibroin solution.
3. And (2) filling the bombyx mori silk fibroin solution into a dialysis bag, wherein the wall of the dialysis bag is a semipermeable membrane, the molecular weight cut-off is 14kDa, placing the dialysis bag filled with the bombyx mori silk fibroin solution into a container filled with deionized water, replacing the water in the container with new deionized water or pure water every 2 hours, and continuously dialyzing for 3 days to obtain the purified bombyx mori silk fibroin aqueous solution. Concentrating by using a rotary evaporator, and adjusting the mass fraction of the dialyzed silk fibroin aqueous solution to be 8%.
4. Adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide accounting for 30-50% of the mass of the silk fibroin and N-hydroxysuccinimide accounting for 15-25% of the mass of the silk fibroin into the silk fibroin aqueous solution, adding 2-morpholine ethanesulfonic acid with the final concentration of 0.05M, stirring in an ice bath for 10-30 minutes, and then pouring into a flat polystyrene plate to be frozen at-80-20 ℃ for 2-24 hours.
5. And (3) taking out the frozen fibroin material, freeze-drying the fibroin material in a freeze dryer, then placing the fibroin material in a container filled with deionized water, soaking for 24 hours, replacing the deionized water in the container every 2 hours, taking out the fibroin material, and freeze-drying the fibroin material again to obtain the fibroin porous material.
6. Soaking the silk fibroin porous material in an aqueous solution containing CGRP with the final concentration of 10nM, wherein the solution is just soaked in the silk fibroin material, placing the silk fibroin porous material at 4 ℃ for 6-10 hours, squeezing and collecting the solution, and air-drying the material at room temperature (25 ℃) to obtain the CGRP-loaded functional silk fibroin porous material.
7. The CGRP content which does not react with silk fibroin in the collected solution is determined by adopting enzyme-linked immunosorbent assay and ultraviolet absorption spectrometry, and the maximum loading of the CGRP in the functional silk fibroin porous material is obtained by conversion calculation.
8. The prepared functional fibroin porous material is cut into small wafers or small squares with proper sizes, the hemolytic performance of the fibroin material is determined according to a hemolytic rate test method, fresh blood diluted by normal saline is taken to be in dynamic contact with the fibroin material, and the hemolytic rate of the functional fibroin porous material is determined to be less than 0.4 percent and completely meets the standard (0-2 percent) of a non-hemolytic material.
Example 2:
the embodiment shows a preparation method of a functional fibroin porous material, which comprises the following steps:
1. the method comprises the steps of putting raw silkworms or cocoon shells of silkworms into a sodium carbonate aqueous solution with the concentration of 0.2% according to the bath ratio of 1:50(g/mL), processing for three times at the temperature of 98-100 ℃ for 30 minutes each time, then fully cleaning the silks with deionized water, loosening the silks, and drying the silks in an oven at the temperature of 60 ℃ to obtain the degummed silk fibroin fibers of the silkworms.
2. Weighing degummed fibroin, dissolving in 9.3M lithium bromide water solution according to a bath ratio of 1:10(g/mL), and treating at 65 deg.C until fibroin fiber is completely dissolved to obtain domestic silkworm fibroin solution.
3. And (2) filling the bombyx mori silk fibroin solution into a dialysis bag, wherein the wall of the dialysis bag is a semipermeable membrane, the molecular weight cut-off is 14kDa, placing the dialysis bag filled with the bombyx mori silk fibroin solution into a container filled with deionized water, replacing the water in the container with new deionized water or pure water every 2 hours, and continuously dialyzing for 3 days to obtain the purified bombyx mori silk fibroin aqueous solution. Concentrating by using a rotary evaporator, and adjusting the mass fraction of the dialyzed silk fibroin aqueous solution to be 8%.
4. Adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide accounting for 30-50% of the mass of the silk fibroin and N-hydroxysuccinimide accounting for 15-25% of the mass of the silk fibroin into the silk fibroin aqueous solution, adding 2-morpholine ethanesulfonic acid with the final concentration of 0.05M, stirring in an ice bath for 10-30 minutes, and then pouring into a flat polystyrene plate to be frozen at-80-20 ℃ for 2-24 hours.
5. And (3) taking out the frozen fibroin material, freeze-drying the fibroin material in a freeze dryer, then placing the fibroin material in a container filled with deionized water, soaking for 24 hours, replacing the deionized water in the container every 2 hours, taking out the fibroin material, and freeze-drying the fibroin material again to obtain the fibroin porous material.
6. Soaking the silk fibroin porous material in an aqueous solution containing CGRP with the final concentration of 50nM, wherein the solution is just soaked in the silk fibroin material, placing the silk fibroin porous material at 4 ℃ for 6-10 hours, squeezing and collecting the solution, and air-drying the material at room temperature (25 ℃) to obtain the CGRP-loaded functional silk fibroin porous material.
7. The CGRP content which does not react with silk fibroin in the collected solution is determined by adopting enzyme-linked immunosorbent assay and ultraviolet absorption spectrometry, and the maximum loading of the CGRP in the functional silk fibroin porous material is obtained by conversion calculation.
8. The prepared functional fibroin porous material is cut into small wafers or small squares with proper sizes, the hemolytic performance of the fibroin material is determined according to a hemolytic rate test method, fresh blood diluted by normal saline is taken to be in dynamic contact with the fibroin material, and the hemolytic rate of the functional fibroin porous material is determined to be less than 0.4 percent and completely meets the standard (0-2 percent) of a non-hemolytic material.
Example 3:
the embodiment shows a preparation method of a functional fibroin porous material, which comprises the following steps:
1. the method comprises the steps of putting raw silkworms or cocoon shells of silkworms into a sodium carbonate aqueous solution with the concentration of 0.2% according to the bath ratio of 1:50(g/mL), processing for three times at the temperature of 98-100 ℃ for 30 minutes each time, then fully cleaning the silks with deionized water, loosening the silks, and drying the silks in an oven at the temperature of 60 ℃ to obtain the degummed silk fibroin fibers of the silkworms.
2. Weighing degummed fibroin, dissolving in 9.3M lithium bromide water solution according to a bath ratio of 1:10(g/mL), and treating at 65 deg.C until fibroin fiber is completely dissolved to obtain domestic silkworm fibroin solution.
3. And (2) filling the bombyx mori silk fibroin solution into a dialysis bag, wherein the wall of the dialysis bag is a semipermeable membrane, the molecular weight cut-off is 14kDa, placing the dialysis bag filled with the bombyx mori silk fibroin solution into a container filled with deionized water, replacing the water in the container with new deionized water or pure water every 2 hours, and continuously dialyzing for 3 days to obtain the purified bombyx mori silk fibroin aqueous solution. Concentrating by using a rotary evaporator, and adjusting the mass fraction of the dialyzed silk fibroin aqueous solution to be 8%.
4. Adding adipic acid with a mass ratio of 100:0.5 to the silk fibroin into the silk fibroin aqueous solution, then respectively adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 15-25% N-hydroxysuccinimide accounting for 30-50% of the mass of the silk fibroin, then adding 2-morpholine ethanesulfonic acid with a final concentration of 0.05M, stirring for 10-30 minutes in an ice bath, and then pouring into a flat polystyrene flat dish to freeze for 2-24 hours at-80 to-20 ℃.
5. And (3) taking out the frozen fibroin material, freeze-drying the fibroin material in a freeze dryer, then placing the fibroin material in a container filled with deionized water, soaking for 24 hours, replacing the deionized water in the container every 2 hours, taking out the fibroin material, and freeze-drying the fibroin material again to obtain the modified fibroin porous material.
6. And (2) soaking the modified silk fibroin porous material in an aqueous solution containing CGRP with the final concentration of 10nM, wherein the solution is preferably just soaked in the silk fibroin material, then placing at 4 ℃ for 6-10 hours, squeezing and collecting the solution, and air-drying the material at room temperature (<25 ℃) to obtain the CGRP-loaded functional silk fibroin porous material.
7. The CGRP content which does not react with silk fibroin in the collected solution is measured by adopting enzyme-linked immunosorbent assay and ultraviolet absorption spectrometry, and the loading amount of the CGRP in the functional silk fibroin porous material obtained by conversion calculation is 1.3 times of that in the example 1.
8. The prepared functional fibroin porous material is cut into small wafers or small squares with proper sizes, the hemolytic performance of the fibroin material is determined according to a hemolytic rate test method, fresh blood diluted by normal saline is taken to be in dynamic contact with the fibroin material, and the hemolytic rate of the functional fibroin porous material is determined to be less than 0.4 percent and completely meets the standard (0-2 percent) of a non-hemolytic material.
Example 4
The embodiment shows a preparation method of a functional fibroin porous material, which comprises the following steps:
1. the method comprises the steps of putting raw silkworms or cocoon shells of silkworms into a sodium carbonate aqueous solution with the concentration of 0.2% according to the bath ratio of 1:50(g/mL), processing for three times at the temperature of 98-100 ℃ for 30 minutes each time, then fully cleaning the silks with deionized water, loosening the silks, and drying the silks in an oven at the temperature of 60 ℃ to obtain the degummed silk fibroin fibers of the silkworms.
2. Weighing degummed fibroin, dissolving in 9.3M lithium bromide water solution according to a bath ratio of 1:10(g/mL), and treating at 65 deg.C until fibroin fiber is completely dissolved to obtain domestic silkworm fibroin solution.
3. And (2) filling the bombyx mori silk fibroin solution into a dialysis bag, wherein the wall of the dialysis bag is a semipermeable membrane, the molecular weight cut-off is 14kDa, placing the dialysis bag filled with the bombyx mori silk fibroin solution into a container filled with deionized water, replacing the water in the container with new deionized water or pure water every 2 hours, and continuously dialyzing for 3 days to obtain the purified bombyx mori silk fibroin aqueous solution. Concentrating by using a rotary evaporator, and adjusting the mass fraction of the dialyzed silk fibroin aqueous solution to be 8%.
4. Adding adipic acid with a mass ratio of 100:0.5 to the silk fibroin into the silk fibroin aqueous solution, then respectively adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 15-25% N-hydroxysuccinimide accounting for 30-50% of the mass of the silk fibroin, then adding 2-morpholine ethanesulfonic acid with a final concentration of 0.05M, stirring for 10-30 minutes in an ice bath, and then pouring into a flat polystyrene flat dish to freeze for 2-24 hours at-80 to-20 ℃.
5. And (3) taking out the frozen fibroin material, freeze-drying the fibroin material in a freeze dryer, then placing the fibroin material in a container filled with deionized water, soaking for 24 hours, replacing the deionized water in the container every 2 hours, taking out the fibroin material, and freeze-drying the fibroin material again to obtain the modified fibroin porous material.
6. And (2) soaking the modified silk fibroin porous material in an aqueous solution containing CGRP with the final concentration of 50nM, wherein the solution is preferably just soaked in the silk fibroin material, then placing at 4 ℃ for 6-10 hours, squeezing and collecting the solution, and air-drying the material at room temperature (<25 ℃) to obtain the CGRP-loaded functional silk fibroin porous material.
7. The CGRP content which does not react with silk fibroin in the collected solution is measured by adopting enzyme-linked immunosorbent assay and ultraviolet absorption spectrometry, and the loading amount of the CGRP in the functional silk fibroin porous material obtained by conversion calculation is 4 times of that of the embodiment 1 and is about 2.7 times of that of the embodiment 2.
8. The prepared functional fibroin porous material is cut into small wafers or small squares with proper sizes, the hemolytic performance of the fibroin material is determined according to a hemolytic rate test method, fresh blood diluted by normal saline is taken to be in dynamic contact with the fibroin material, and the hemolytic rate of the functional fibroin porous material is determined to be less than 0.4 percent and completely meets the standard (0-2 percent) of a non-hemolytic material.
Example 5
The embodiment shows a preparation method of a functional fibroin porous material, which comprises the following steps:
1. the method comprises the steps of putting raw silkworms or cocoon shells of silkworms into a sodium carbonate aqueous solution with the concentration of 0.2% according to the bath ratio of 1:50(g/mL), processing for three times at the temperature of 98-100 ℃ for 30 minutes each time, then fully cleaning the silks with deionized water, loosening the silks, and drying the silks in an oven at the temperature of 60 ℃ to obtain the degummed silk fibroin fibers of the silkworms.
2. Weighing degummed fibroin, dissolving in 9.3M lithium bromide water solution according to a bath ratio of 1:10(g/mL), and treating at 65 deg.C until fibroin fiber is completely dissolved to obtain domestic silkworm fibroin solution.
3. And (2) filling the bombyx mori silk fibroin solution into a dialysis bag, wherein the wall of the dialysis bag is a semipermeable membrane, the molecular weight cut-off is 14kDa, placing the dialysis bag filled with the bombyx mori silk fibroin solution into a container filled with deionized water, replacing the water in the container with new deionized water or pure water every 2 hours, and continuously dialyzing for 3 days to obtain the purified bombyx mori silk fibroin aqueous solution. Concentrating by using a rotary evaporator, and adjusting the mass fraction of the dialyzed silk fibroin aqueous solution to be 8%.
4. Adding adipic acid with a mass ratio of 100:2.0 to the silk fibroin into the silk fibroin aqueous solution, then respectively adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 15-25% N-hydroxysuccinimide accounting for 30-50% of the mass of the silk fibroin, then adding 2-morpholine ethanesulfonic acid with a final concentration of 0.05M, stirring for 10-30 minutes in an ice bath, and then pouring into a flat polystyrene flat dish to freeze for 2-24 hours at-80 to-20 ℃.
5. And (3) taking out the frozen fibroin material, freeze-drying the fibroin material in a freeze dryer, then placing the fibroin material in a container filled with deionized water, soaking for 24 hours, replacing the deionized water in the container every 2 hours, taking out the fibroin material, and freeze-drying the fibroin material again to obtain the modified fibroin porous material.
6. And (2) soaking the modified silk fibroin porous material in an aqueous solution containing CGRP with the final concentration of 50nM, wherein the solution is preferably just soaked in the silk fibroin material, then placing at 4 ℃ for 6-10 hours, squeezing and collecting the solution, and air-drying the material at room temperature (<25 ℃) to obtain the CGRP-loaded functional silk fibroin porous material.
7. The CGRP content which does not react with silk fibroin in the collected solution is determined by adopting enzyme-linked immunosorbent assay and ultraviolet absorption spectrometry, and the loading amount of the CGRP in the functional silk fibroin porous material obtained by conversion calculation is 2 times of that in the example 4.
8. The prepared functional fibroin porous material is cut into small wafers or small squares with proper sizes, the hemolytic performance of the fibroin material is determined according to a hemolytic rate test method, fresh blood diluted by normal saline is taken to be in dynamic contact with the fibroin material, and the hemolytic rate of the functional fibroin porous material is determined to be less than 0.4 percent and completely meets the standard (0-2 percent) of a non-hemolytic material.
Example 6
The embodiment shows a preparation method of a functional fibroin porous material, which comprises the following steps:
1. the method comprises the steps of putting raw silkworms or cocoon shells of silkworms into a sodium carbonate aqueous solution with the concentration of 0.2% according to the bath ratio of 1:50(g/mL), processing for three times at the temperature of 98-100 ℃ for 30 minutes each time, then fully cleaning the silks with deionized water, loosening the silks, and drying the silks in an oven at the temperature of 60 ℃ to obtain the degummed silk fibroin fibers of the silkworms.
2. Weighing degummed fibroin, dissolving in 9.3M lithium bromide water solution according to a bath ratio of 1:10(g/mL), and treating at 65 deg.C until fibroin fiber is completely dissolved to obtain domestic silkworm fibroin solution.
3. And (2) filling the bombyx mori silk fibroin solution into a dialysis bag, wherein the wall of the dialysis bag is a semipermeable membrane, the molecular weight cut-off is 14kDa, placing the dialysis bag filled with the bombyx mori silk fibroin solution into a container filled with deionized water, replacing the water in the container with new deionized water or pure water every 2 hours, and continuously dialyzing for 3 days to obtain the purified bombyx mori silk fibroin aqueous solution. Concentrating by using a rotary evaporator, and adjusting the mass fraction of the dialyzed silk fibroin aqueous solution to be 8%.
4. Adding adipic acid with a mass ratio of 100:2.0 to the silk fibroin into the silk fibroin aqueous solution, then respectively adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 15-25% N-hydroxysuccinimide accounting for 30-50% of the mass of the silk fibroin, then adding 2-morpholine ethanesulfonic acid with a final concentration of 0.05M, stirring for 10-30 minutes in an ice bath, and then pouring into a flat polystyrene flat dish to freeze for 2-24 hours at-80 to-20 ℃.
5. And (3) taking out the frozen fibroin material, freeze-drying the fibroin material in a freeze dryer, then placing the fibroin material in a container filled with deionized water, soaking for 24 hours, replacing the deionized water in the container every 2 hours, taking out the fibroin material, and freeze-drying the fibroin material again to obtain the modified fibroin porous material.
6. And (2) soaking the modified silk fibroin porous material in an aqueous solution containing CGRP with the final concentration of 200nM, wherein the solution is preferably just soaked in the silk fibroin material, then placing at 4 ℃ for 6-10 hours, squeezing and collecting the solution, and air-drying the material at room temperature (<25 ℃) to obtain the CGRP-loaded functional silk fibroin porous material.
7. The CGRP content of the collected solution which is not reacted with silk fibroin is measured by adopting enzyme-linked immunosorbent assay and ultraviolet absorption spectrometry, and the loading amount of the CGRP in the functional silk fibroin porous material is obtained by conversion calculation and is about 2.5 times of that of the embodiment 5, and the loading amount is about 5 times of that of the embodiment 4.
8. The prepared functional fibroin porous material is cut into small wafers or small squares with proper sizes, the hemolytic performance of the fibroin material is determined according to a hemolytic rate test method, fresh blood diluted by normal saline is taken to be in dynamic contact with the fibroin material, and the hemolytic rate of the functional fibroin porous material is determined to be less than 0.4 percent and completely meets the standard (0-2 percent) of a non-hemolytic material.
Example 7
The embodiment shows a preparation method of a functional silk fibroin membrane, which comprises the following steps:
1. the method comprises the steps of putting raw silkworms or cocoon shells of silkworms into a sodium carbonate aqueous solution with the concentration of 0.2% according to the bath ratio of 1:50(g/mL), processing for three times at the temperature of 98-100 ℃ for 30 minutes each time, then fully cleaning the silks with deionized water, loosening the silks, and drying the silks in an oven at the temperature of 60 ℃ to obtain the degummed silk fibroin fibers of the silkworms.
2. Weighing degummed fibroin, dissolving in 9.3M lithium bromide water solution according to a bath ratio of 1:10(g/mL), and treating at 65 deg.C until fibroin fiber is completely dissolved to obtain domestic silkworm fibroin solution.
3. And (2) filling the bombyx mori silk fibroin solution into a dialysis bag, wherein the wall of the dialysis bag is a semipermeable membrane, the molecular weight cut-off is 14kDa, placing the dialysis bag filled with the bombyx mori silk fibroin solution into a container filled with deionized water, replacing the water in the container with new deionized water or pure water every 2 hours, and continuously dialyzing for 3 days to obtain the purified bombyx mori silk fibroin aqueous solution. Concentrating by adopting a rotary evaporator, and adjusting the mass fraction of the silk fibroin after dialysis to be 5%.
4. And (2) respectively adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide accounting for 30-50% of the mass of the silk fibroin and N-hydroxysuccinimide accounting for 15-25% of the mass of the silk fibroin into the silk fibroin aqueous solution, adding 2-morpholine ethanesulfonic acid with the final concentration of 0.05M, stirring in an ice bath for 10-30 minutes, and pouring into a flat polystyrene plate to dry at 40-60 ℃.
5. And taking out the dried film, putting the film into a container filled with deionized water, soaking for 2 days, replacing the deionized water in the container every 2-4 hours, taking out the film after 2 days, and air-drying to obtain the fibroin film.
6. The surface charge of the silk fibroin film is measured to be-14.3 mV by adopting a solid surface Zeta potentiometer.
7. Placing the silk fibroin film back into a polystyrene plate, spreading, and adding 100ng/cm2The aqueous solution of CGRP with positive charge is placed at 4 ℃ for 6 to 10 hours, then taken out and collected, and the material is cooled to room temperature (<And air-drying at 25 ℃ to obtain the CGRP-loaded functional silk fibroin film.
8. The CGRP content which does not react with silk fibroin in the collected solution is determined by adopting enzyme-linked immunosorbent assay and ultraviolet absorption spectrometry, and the maximum loading of the CGRP in the functional silk fibroin membrane is obtained by conversion calculation.
9. The prepared functional silk fibroin membrane is cut into small wafers or small squares with proper sizes, the hemolytic performance of the silk fibroin material is determined according to a hemolytic rate test method, fresh blood diluted by normal saline is taken to be in dynamic contact with the silk fibroin material, and the hemolytic rate of the functional silk fibroin membrane is determined to be less than 0.4%, and completely accords with the standard (0-2%) of a non-hemolytic material.
Example 8
The embodiment shows a preparation method of a functional silk fibroin membrane, which comprises the following steps:
1. the method comprises the steps of putting raw silkworms or cocoon shells of silkworms into a sodium carbonate aqueous solution with the concentration of 0.2% according to the bath ratio of 1:50(g/mL), processing for three times at the temperature of 98-100 ℃ for 30 minutes each time, then fully cleaning the silks with deionized water, loosening the silks, and drying the silks in an oven at the temperature of 60 ℃ to obtain the degummed silk fibroin fibers of the silkworms.
2. Weighing degummed fibroin, dissolving in 9.3M lithium bromide water solution according to a bath ratio of 1:10(g/mL), and treating at 65 deg.C until fibroin fiber is completely dissolved to obtain domestic silkworm fibroin solution.
3. And (2) filling the bombyx mori silk fibroin solution into a dialysis bag, wherein the wall of the dialysis bag is a semipermeable membrane, the molecular weight cut-off is 14kDa, placing the dialysis bag filled with the bombyx mori silk fibroin solution into a container filled with deionized water, replacing the water in the container with new deionized water or pure water every 2 hours, and continuously dialyzing for 3 days to obtain the purified bombyx mori silk fibroin aqueous solution. Concentrating by adopting a rotary evaporator, and adjusting the mass fraction of the silk fibroin after dialysis to be 5%.
4. Adding adipic acid with a mass ratio of 100:1.0 to the fibroin into the silk fibroin aqueous solution, then respectively adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 15-25% N-hydroxysuccinimide with a mass ratio of 30-50% of the mass of the silk fibroin, then adding 2-morpholine ethanesulfonic acid with a final concentration of 0.05M, stirring for 10-30 minutes in an ice bath, and then pouring into a flat polystyrene flat dish to dry at 40-60 ℃.
5. And taking out the dried film, putting the film into a container filled with deionized water, soaking for 2 days, replacing the deionized water in the container every 2-4 hours, taking out the film after 2 days, and air-drying to obtain the modified fibroin film.
6. The surface charge of the modified silk fibroin film measured by a solid surface Zeta potentiometer is-18.4 mV, and the negative charge on the surface of the silk fibroin film is obviously increased.
7. The modified silk fibroin film is put back into a polystyrene plate and is paved, and 100ng/cm is added2The aqueous solution of CGRP with positive charge is placed at 4 ℃ for 6 to 10 hours, then taken out and collected, and the material is cooled to room temperature (<And air-drying at 25 ℃ to obtain the CGRP-loaded functional silk fibroin film.
8. The content of CGRP which does not react with silk fibroin in the collected solution is determined by adopting enzyme-linked immunosorbent assay and ultraviolet absorption spectrometry, and the maximum loading of CGRP in the functional silk fibroin membrane is obtained by conversion calculation and is 1.4 times of that in the example 7.
9. The prepared functional silk fibroin membrane is cut into small wafers or small squares with proper sizes, the hemolytic performance of the silk fibroin material is determined according to a hemolytic rate test method, fresh blood diluted by normal saline is taken to be in dynamic contact with the silk fibroin material, and the hemolytic rate of the functional silk fibroin membrane is determined to be less than 0.4%, and completely accords with the standard (0-2%) of a non-hemolytic material.
Example 9
The embodiment shows a preparation method of a functional silk fibroin membrane, which comprises the following steps:
1. the method comprises the steps of putting raw silkworms or cocoon shells of silkworms into a sodium carbonate aqueous solution with the concentration of 0.2% according to the bath ratio of 1:50(g/mL), processing for three times at the temperature of 98-100 ℃ for 30 minutes each time, then fully cleaning the silks with deionized water, loosening the silks, and drying the silks in an oven at the temperature of 60 ℃ to obtain the degummed silk fibroin fibers of the silkworms.
2. Weighing degummed fibroin, dissolving in 9.3M lithium bromide water solution according to a bath ratio of 1:10(g/mL), and treating at 65 deg.C until fibroin fiber is completely dissolved to obtain domestic silkworm fibroin solution.
3. And (2) filling the bombyx mori silk fibroin solution into a dialysis bag, wherein the wall of the dialysis bag is a semipermeable membrane, the molecular weight cut-off is 14kDa, placing the dialysis bag filled with the bombyx mori silk fibroin solution into a container filled with deionized water, replacing the water in the container with new deionized water or pure water every 2 hours, and continuously dialyzing for 3 days to obtain the purified bombyx mori silk fibroin aqueous solution. Concentrating by adopting a rotary evaporator, and adjusting the mass fraction of the silk fibroin after dialysis to be 5%.
4. Adding adipic acid with a mass ratio of 100:1.0 to the fibroin into the silk fibroin aqueous solution, then respectively adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 15-25% N-hydroxysuccinimide with a mass ratio of 30-50% of the mass of the silk fibroin, then adding 2-morpholine ethanesulfonic acid with a final concentration of 0.05M, stirring for 10-30 minutes in an ice bath, and then pouring into a flat polystyrene flat dish to dry at 40-60 ℃.
5. And taking out the dried film, putting the film into a container filled with deionized water, soaking for 2 days, replacing the deionized water in the container every 2-4 hours, taking out the film after 2 days, and air-drying to obtain the modified fibroin film.
6. The surface charge of the modified silk fibroin film measured by a solid surface Zeta potentiometer is-18.4 mV, and the negative charge on the surface of the silk fibroin film is obviously increased.
7. The modified silk fibroin film is put back into a polystyrene plate and is paved, and 200ng/cm is added2The aqueous solution of CGRP with positive charge is placed at 4 ℃ for 6 to 10 hours, then taken out and collected, and the material is cooled to room temperature (<And air-drying at 25 ℃ to obtain the CGRP-loaded functional silk fibroin film.
8. The content of CGRP which does not react with silk fibroin in the collected solution is determined by adopting enzyme-linked immunosorbent assay and ultraviolet absorption spectrometry, and the maximum loading of CGRP in the functional silk fibroin membrane is obtained by conversion calculation and is 1.25 times of that in the example 8.
9. The prepared functional silk fibroin membrane is cut into small wafers or small squares with proper sizes, the hemolytic performance of the silk fibroin material is determined according to a hemolytic rate test method, fresh blood diluted by normal saline is taken to be in dynamic contact with the silk fibroin material, and the hemolytic rate of the functional silk fibroin membrane is determined to be less than 0.4%, and completely accords with the standard (0-2%) of a non-hemolytic material.
Example 10
The embodiment shows a preparation method of a functional silk fibroin membrane, which comprises the following steps:
1. the method comprises the steps of putting raw silkworms or cocoon shells of silkworms into a sodium carbonate aqueous solution with the concentration of 0.2% according to the bath ratio of 1:50(g/mL), processing for three times at the temperature of 98-100 ℃ for 30 minutes each time, then fully cleaning the silks with deionized water, loosening the silks, and drying the silks in an oven at the temperature of 60 ℃ to obtain the degummed silk fibroin fibers of the silkworms.
2. Weighing degummed fibroin, dissolving in 9.3M lithium bromide water solution according to a bath ratio of 1:10(g/mL), and treating at 65 deg.C until fibroin fiber is completely dissolved to obtain domestic silkworm fibroin solution.
3. And (2) filling the bombyx mori silk fibroin solution into a dialysis bag, wherein the wall of the dialysis bag is a semipermeable membrane, the molecular weight cut-off is 14kDa, placing the dialysis bag filled with the bombyx mori silk fibroin solution into a container filled with deionized water, replacing the water in the container with new deionized water or pure water every 2 hours, and continuously dialyzing for 3 days to obtain the purified bombyx mori silk fibroin aqueous solution. Concentrating by adopting a rotary evaporator, and adjusting the mass fraction of the silk fibroin after dialysis to be 5%.
4. Adding adipic acid with a mass ratio of 100:2.0 to the silk fibroin into the silk fibroin aqueous solution, then respectively adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 15-25% N-hydroxysuccinimide with a mass ratio of 30-50% of the silk fibroin, then adding 2-morpholinoethanesulfonic acid with a final concentration of 0.05M, stirring for 10-30 minutes in an ice bath, and then pouring into a flat polystyrene plate to dry at 40-60 ℃.
5. And taking out the dried film, putting the film into a container filled with deionized water, soaking for 2 days, replacing the deionized water in the container every 2-4 hours, taking out the film after 2 days, and air-drying to obtain the modified fibroin film.
6. The surface charge of the modified silk fibroin film measured by a solid surface Zeta potentiometer is-28 mV, and the negative charge on the surface of the silk fibroin film is obviously increased.
7. The modified silk fibroin film is put back into a polystyrene plate and is paved, and 200ng/cm is added2The aqueous solution of CGRP with positive charge is placed at 4 ℃ for 6 to 10 hours, then taken out and collected, and the material is cooled to room temperature (<And air-drying at 25 ℃ to obtain the CGRP-loaded functional silk fibroin film.
8. The content of CGRP which does not react with silk fibroin in the collected solution is determined by adopting enzyme-linked immunosorbent assay and ultraviolet absorption spectrometry, and the maximum loading of CGRP in the functional silk fibroin membrane is obtained by conversion calculation and is 1.4 times of that in the example 9.
9. The prepared functional silk fibroin membrane is cut into small wafers or small squares with proper sizes, the hemolytic performance of the silk fibroin material is determined according to a hemolytic rate test method, fresh blood diluted by normal saline is taken to be in dynamic contact with the silk fibroin material, and the hemolytic rate of the functional silk fibroin membrane is determined to be less than 0.4%, and completely accords with the standard (0-2%) of a non-hemolytic material.
Example 11
The embodiment shows a preparation method of a functional silk fibroin membrane, which comprises the following steps:
1. the method comprises the steps of putting raw silkworms or cocoon shells of silkworms into a sodium carbonate aqueous solution with the concentration of 0.2% according to the bath ratio of 1:50(g/mL), processing for three times at the temperature of 98-100 ℃ for 30 minutes each time, then fully cleaning the silks with deionized water, loosening the silks, and drying the silks in an oven at the temperature of 60 ℃ to obtain the degummed silk fibroin fibers of the silkworms.
2. Weighing degummed fibroin, dissolving in 9.3M lithium bromide water solution according to a bath ratio of 1:10(g/mL), and treating at 65 deg.C until fibroin fiber is completely dissolved to obtain domestic silkworm fibroin solution.
3. And (2) filling the bombyx mori silk fibroin solution into a dialysis bag, wherein the wall of the dialysis bag is a semipermeable membrane, the molecular weight cut-off is 14kDa, placing the dialysis bag filled with the bombyx mori silk fibroin solution into a container filled with deionized water, replacing the water in the container with new deionized water or pure water every 2 hours, and continuously dialyzing for 3 days to obtain the purified bombyx mori silk fibroin aqueous solution. Concentrating by adopting a rotary evaporator, and adjusting the mass fraction of the silk fibroin after dialysis to be 5%.
4. Adding adipic acid with a mass ratio of 100:2.0 to the silk fibroin into the silk fibroin aqueous solution, then respectively adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 15-25% N-hydroxysuccinimide with a mass ratio of 30-50% of the silk fibroin, then adding 2-morpholinoethanesulfonic acid with a final concentration of 0.05M, stirring for 10-30 minutes in an ice bath, and then pouring into a flat polystyrene plate to dry at 40-60 ℃.
5. And taking out the dried film, putting the film into a container filled with deionized water, soaking for 2 days, replacing the deionized water in the container every 2-4 hours, taking out the film after 2 days, and air-drying to obtain the modified fibroin film.
6. The surface charge of the modified silk fibroin film measured by a solid surface Zeta potentiometer is-28 mV, and the negative charge on the surface of the silk fibroin film is obviously increased.
7. The modified silk fibroin film is put back into a polystyrene plate and is paved, and 400ng/cm is added2The aqueous solution of CGRP with positive charge is placed at 4 ℃ for 6 to 10 hours, then taken out and collected, and the material is cooled to room temperature (<And air-drying at 25 ℃ to obtain the CGRP-loaded functional silk fibroin film.
8. The content of CGRP which does not react with silk fibroin in the collected solution is measured by adopting enzyme-linked immunosorbent assay and ultraviolet absorption spectrometry, and the maximum loading of CGRP in the functional silk fibroin membrane is obtained by conversion calculation and is 1.44 times of that in the example 10.
9. The prepared functional silk fibroin membrane is cut into small wafers or small squares with proper sizes, the hemolytic performance of the silk fibroin material is determined according to a hemolytic rate test method, fresh blood diluted by normal saline is taken to be in dynamic contact with the silk fibroin material, and the hemolytic rate of the functional silk fibroin membrane is determined to be less than 0.4%, and completely accords with the standard (0-2%) of a non-hemolytic material.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims (5)

1. A method for preparing a functional fibroin porous material or a functional fibroin membrane is characterized by comprising the following steps:
(1) preparing degummed silkworm fibroin fibers: placing silkworm silk or cocoon shells into a sodium carbonate aqueous solution, heating, cleaning, loosening, and drying to obtain degummed silkworm fibroin fibers;
(2) preparing a silkworm fibroin dissolving solution: completely dissolving the degummed bombyx mori silk fibroin fibers in a lithium bromide aqueous solution to obtain a silk fibroin dissolving solution;
(3) preparing a purified silkworm silk fibroin aqueous solution: filling the fibroin dissolving solution into a dialysis bag, then placing the dialysis bag into a container filled with deionized water, replacing the liquid in the container with new deionized water or pure water every 2 hours, continuously dialyzing for 3 days, and concentrating to obtain a purified silkworm fibroin aqueous solution;
(4) preparing a modified fibroin porous material or a modified fibroin membrane: adding adipic acid into the purified silkworm fibroin aqueous solution, wherein the mass ratio of the purified silkworm fibroin to the adipic acid is 100: 1-20, then adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide, the adding amount of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is 30-50% of the mass of the purified silkworm fibroin, the adding amount of the N-hydroxysuccinimide is 15-25% of the mass of the purified silkworm fibroin, then adding 2-morpholine ethanesulfonic acid, the final concentration of the 2-morpholine ethanesulfonic acid is 0.05M, stirring for 10-30 minutes in an ice bath, freeze-drying or drying, then placing the freeze-dried or dried material into a container containing deionized water, and soaking for 24-48 hours, and replacing the deionized water in the container every 2-4 hours, taking out, and freeze-drying or air-drying again to obtain the modified fibroin porous material or the modified fibroin membrane, wherein the freeze-drying specifically comprises the following steps: pre-freezing for 2-24 hours at the temperature of-80 ℃ to-20 ℃, and then putting the mixture into a freeze dryer for freeze drying, wherein the drying is to pour the mixed solution into a flat polystyrene plate and dry the mixed solution at the temperature of 40-60 ℃;
(5) preparing a functional silk fibroin porous material or a functional silk fibroin film: soaking the modified fibroin porous material in a CGRP aqueous solution with positive charges, wherein the final concentration of the CGRP aqueous solution is 10-1000 nM, or adding the CGRP aqueous solution with positive charges on the modified fibroin membrane, and the concentration of the CGRP aqueous solution is 50-1000 ng/cm2Standing, collecting the solution, and air-drying the material at a temperature of less than 25 ℃ to obtain the functional fibroin porous material or the functional fibroin membrane.
2. The method for preparing the functional silk fibroin porous material or the functional silk fibroin membrane according to claim 1, characterized in that: the step (1) of putting the silkworm silk or cocoon shells into the sodium carbonate aqueous solution for heating treatment refers to putting the silkworm silk or cocoon shells into the sodium carbonate aqueous solution with the mass concentration of 0.2-0.8% according to the bath ratio of 1g to 50mL, wherein the heating treatment refers to treatment for 2-3 times at the temperature of 98-100 ℃ for 30 minutes each time, and the drying refers to drying in an oven at the temperature of 60 ℃.
3. The method for preparing the functional silk fibroin porous material or the functional silk fibroin membrane according to claim 1, characterized in that: the step (2) of completely dissolving the degummed bombyx mori silk fibroin fibers in the lithium bromide aqueous solution means that the degummed bombyx mori silk fibroin fibers are weighed and dissolved in 9.3M lithium bromide aqueous solution according to the bath ratio of 1g to 10mL, and the solution is treated at the temperature of 65 ℃ until the silk fibroin fibers are completely dissolved.
4. The method for preparing the functional silk fibroin porous material or the functional silk fibroin membrane according to claim 1, characterized in that: in the step (3), the dialysis bag is a semipermeable membrane, the molecular weight cutoff is 10-14 kDa, and the concentration is specifically performed by adopting a rotary evaporator, so that the mass fraction of the dialyzed bombyx mori silk fibroin aqueous solution is 5-15%.
5. The method for preparing the functional silk fibroin porous material or the functional silk fibroin membrane according to claim 1, characterized in that: and (5) standing for 6-10 hours at the temperature of 4 ℃.
CN201910304083.6A 2019-04-16 2019-04-16 Functional silk fibroin porous material or functional silk fibroin membrane and preparation method thereof Active CN110041557B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910304083.6A CN110041557B (en) 2019-04-16 2019-04-16 Functional silk fibroin porous material or functional silk fibroin membrane and preparation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910304083.6A CN110041557B (en) 2019-04-16 2019-04-16 Functional silk fibroin porous material or functional silk fibroin membrane and preparation method thereof

Publications (2)

Publication Number Publication Date
CN110041557A CN110041557A (en) 2019-07-23
CN110041557B true CN110041557B (en) 2021-08-31

Family

ID=67277366

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910304083.6A Active CN110041557B (en) 2019-04-16 2019-04-16 Functional silk fibroin porous material or functional silk fibroin membrane and preparation method thereof

Country Status (1)

Country Link
CN (1) CN110041557B (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105295080A (en) * 2015-10-27 2016-02-03 苏州大学 Method for preparing functional silk fibroin protein membrane beneficial to cell adhesion

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105295080A (en) * 2015-10-27 2016-02-03 苏州大学 Method for preparing functional silk fibroin protein membrane beneficial to cell adhesion

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Vascular Cell Co-Culture on Silk Fibroin Matrix;Fangfang Tu et al.;《Polymers》;20180101;第10卷;10010039 *
基于基因工程多肽物理水凝胶的制备及其应用;姚明浩;《中国博士学位论文全文数据库 医药卫生科技辑》;20170815(第08期);E080-15 *
降钙素基因相关肽对培养的血管内皮细胞缺氧再给氧的影响;任雨笙 等;《中国病理生理杂志》;20010228;第17卷(第2期);第101-103页 *

Also Published As

Publication number Publication date
CN110041557A (en) 2019-07-23

Similar Documents

Publication Publication Date Title
CN110025828B (en) Functional silk fibroin porous material or functional silk fibroin film and preparation method thereof
CN110003518B (en) Active silk fibroin porous material or active silk fibroin film and preparation method thereof
Lamboni et al. Silk sericin: A versatile material for tissue engineering and drug delivery
Vasconcelos et al. The use of keratin in biomedical applications
CN110075309B (en) Silk fibroin film with function of regulating and controlling growth of blood vessel cells and preparation method thereof
WO2002034885A1 (en) Sericin-containing material, process for producing the same and method of using the same
CN112480434B (en) Copper ion antibacterial hydrogel and preparation method and application thereof
JP4551761B2 (en) Keratin-derived orthopedic material
CN110038162B (en) Functional silk fibroin material with function of regulating and controlling growth of vascular cells and preparation method thereof
CN105797203A (en) Alginate fiber based collagen sponge dressing and preparation method thereof
CN110384818A (en) A kind of alginate dressing
CN110066418B (en) Active silk fibroin porous material or active silk fibroin membrane and preparation method thereof
CN107708722A (en) Chronic wound treatment is with composition, its preparation method and the chronic wound treatment dressing using it
CN110152055A (en) The functional drug that alginic acid amination derivative/bacteria cellulose nanocomposite gel is constructed is sustained medical dressing
WO2017101020A1 (en) Modified dressing
WO2023056716A1 (en) Silk fibroin composite porous stent and preparation method therefor
CN110041557B (en) Functional silk fibroin porous material or functional silk fibroin membrane and preparation method thereof
Tseomashko et al. New hybrid materials for wound cover dressings
CN115010981B (en) Solid product containing amino indissolvable polymer, preparation method and application thereof
CN109431970B (en) Fiber bundle/polypeptide hydrogel containing double drugs for promoting wound healing and preparation method thereof
CN102008740B (en) Absorbable growth factor composite dressing
JP2955653B2 (en) Silk protein / collagen composite and method for producing the same
CN114524970B (en) Chitosan/silk fibroin sponge loaded with human recombinant bone morphogenetic protein rhBMP-2
AU2021100655A4 (en) POLY γ-GLUTAMIC ACID/CHITOSAN/CALCIUM CITRATE BIOMATERIAL AND PREPARATION METHOD THEREO
KR19990049107A (en) Manufacturing method of polymer electrolyte composite sponge

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
GR01 Patent grant
GR01 Patent grant