CN101412853B - Anti-gelation silk fibroin water solution and preparation thereof - Google Patents
Anti-gelation silk fibroin water solution and preparation thereof Download PDFInfo
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- CN101412853B CN101412853B CN2008102352226A CN200810235222A CN101412853B CN 101412853 B CN101412853 B CN 101412853B CN 2008102352226 A CN2008102352226 A CN 2008102352226A CN 200810235222 A CN200810235222 A CN 200810235222A CN 101412853 B CN101412853 B CN 101412853B
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Abstract
The invention discloses an anti-gelling silk fibroin aqueous solution and a preparation method thereof, which belong to the technical field of natural macromolecular material. The method uses water as a solvent and vitamin C with biocompatibility as an anticoagulant; and the water and the vitamin C have the advantages of innocuity and harmfulness and meet the requirement of development and application of a new-generation biological material. The method utilizes mutual bonding between oxhydryl of a vitamin C molecule and oxhydry of a silk fibroin molecule, weakens the function of a hydrogen bond of the silk fibroin molecule or between several silk fibroin macromolecules in the aqueous solution, obviously improves the stability of the silk fibroin aqueous solution, solves the problem that the regenerated silk fibroin aqueous solution with high concentration is rapidly gelled in the storage process at room temperature, can be widely applied to the field of biological medical material, and meets the requirement of development and application of the new generation biological material.
Description
Technical field
The present invention relates to a kind of silk fibroin water solution and preparation method thereof, particularly a kind of silk fibroin water solution with anti-gel characteristic and preparation method thereof belongs to the natural macromolecular material technical field.
Background technology
Silk is the natural macromolecular material with excellent mechanical property, and its existing higher intensity has stronger toughness again.Silkworm " spinning " carried out at normal temperatures and pressures, and is solvent with water, and environmentally safe meets the trend that environmental protection develops.Silk fibroin is one of component in the silk fiber, as natural biological albumen, except having excellent mechanical property, also has excellent biological compatibility, is subjected to biomaterial circle extensive studies.Support based on silk fibroin has been proved to be and may be widely used aspect the making repair tissue material, and the electrostatic spinning non-woven fabrics is owing to have big special surface zone and the porous three-dimensional structure that helps high-density cells and tissue culture, thereby is to be hopeful one of material forms that is applied to all kinds of organizational projects most.
(Polymer.2003 (44): 841-846) used the solvent of HFA in the document that is entitled as " Preparation of non-woven nanofibers of Bombyx mori silk; Samia Cynthia ricini silk and recombinant hybrid silk witheletrospinning method " as spinning, make the silk fibroin nano-fiber of mean diameter between 100～1000nm, obtained preliminary success; At publication number is in the Chinese invention patent " a kind of composite nano-grade silk fiber product and preparation method thereof " of CN1837435, with the silk fibroin protein solution drying and forming-film, be dissolved in the formic acid then, and and nylon 6 mixing, obtained the non-woven fabrics that nano level silk fibroin/nylon 6 conjugated fibres constitute by electrostatic spinning.Because used solvent in the technique scheme, some easily causes the degraded of silk fibroin, what have has very strong corrodibility, and adopt the organic solvent electrostatic spinning to prepare the silk fibroin non-woven fabrics, may cause dissolvent residual, residual solvent has cytotoxicity may cause the immune response of fiber and surrounding tissue, is very restricted in production application.
The dense silk fibroin water solution that uses water as solvent is the best solution of regenerated silk protein spinning research.Yet the preparation process of regenerated silk fibroin water solution from initial coming unstuck, to obtaining certain density silk fibroin water solution, experience the time in an about week.Gelation can take place under certain condition as a kind of natural protein colloidal sol in regenerated silk protein solution, and this is because silk fibroin molecular is converted into the event of beta sheet conformation by random coil.Therefore, the stability of raising regenerated silk fibroin water solution is the prerequisite of carrying out the research of regenerated silk protein biomedical applications.
(Biological Macromolecules.2003 in being entitled as " The role of formic acid in solution stability and crystallization ofsilk protein polymer " document, 33:203-213), In Chul Um, Hae Yong Kweon etc. has studied the influence of acetate to silk fibroin stability and crystal property, analytical test shows in acetate, the shape of molecule comparatively dense of silk fibroin, therefore, intermolecular intermeshing chance reduces, and the stability of silk fibroin better.But acetate has stronger corrodibility, does not meet the requirement of biomaterial preparation.(synthon .2007 in being entitled as " regenerated silk fibroin water solution Study on Stability ", 13:10-12), Wang Hong, people's results of study such as Zhang Yaopeng show, silk fibroin water solution concentration is high more, stability is poor more, has only just to obtain stability preferably at low temperatures, and the shelf-time is long.
" Gelation of silk fibroin " (SEN I GAKKAISHI.1990,46 (11): 521-524), Hirabayashi etc. think that the formation of micro gel is because formed hydrogen bonded in the silk fibroin molecular or between several silk fibroin macromole.Be entitled as " research of silk fibroin structure---the discussion fibroin forms the mechanism of gel " (Chinese food journal .2001,1 (2): 10-13), Ni Li etc. test the structure of silk extract gel with Raman spectrum, and the result thinks that also hydrogen bond has been brought into play vital role when forming gel.The formation of hydrogen bond is the key that improves silk fibroin stability in the blocking-up silk fibroin molecular or between several silk fibroin macromole.
Summary of the invention
In order to overcome the deficiency that prior art exists, the invention provides a kind of silk fibroin water solution and preparation method thereof with anti-gel property.
To achieve the above object of the invention, the technical solution adopted in the present invention is: a kind of anti-gelation silk fibroin water solution is provided, contain silk fibroin and antithrombotics vitamins C in the aqueous solution, wherein, the mass concentration of silk fibroin is 5～50%, and the ascorbic mass ratio of silk fibroin and antithrombotics is 300:1～10:1.
A kind of preparation method of anti-gelation silk fibroin water solution, adopt following steps:
A. prepare silk fibroin quality concentration and be 5～50% silk fibroin water solution;
B. add the antithrombotics vitamins C in the above-mentioned aqueous solution, the ascorbic mass ratio of silk fibroin and antithrombotics is 300:1～10:1, and constant temperature stirs in the water-bath below 50 ℃, obtains anti-gelation silk fibroin water solution.
Described silk fibroin water solution by natural silk through coming unstuck, obtaining after swelling, dissolving, dialysis, centrifugation and the concentration.
Studies show that the formation of micro gel is because formed hydrogen bonded in the silk fibroin molecular or between several silk fibroin macromole in the silk fibroin water solution, therefore, the formation of blocking-up hydrogen bond is the key that improves silk fibroin water solution stability.The present invention is the mechanism that forms hydrogen bond according to bonding between the hydroxyl, select the many soluble small molecular vitamins Cs of hydroxyl on the molecule, mutual bonding has taken place in hydroxyl on hydroxyl on its molecule and the silk fibroin molecular, make in the aqueous solution in the silk fibroin molecular and intermolecular mutual entanglement is weakened, thereby improved silk fibroin water solution stability at room temperature.
The present invention is directed to high density silk fibroin water solution instability in depositing process under the current room temperature, even the problem of gel also can take place under the storage condition of sealing automatically, employing as antithrombotics, improves silk fibroin water solution stability with the biocompatible substance vitamins C, and its advantage is:
(1) utilizes the mutual bonding that hydroxyl takes place on hydroxyl on the vitamins C molecule and the silk fibroin molecular, weakened in the aqueous solution in the silk fibroin molecular or the effect of hydrogen bond between several silk fibroin macromole, obviously improve the stability of silk fibroin water solution, satisfied the needs of high density regenerated silk fibroin water solution spinning research.
(2) as antithrombotics, water is solvent with biocompatible substance in the present invention, and the silk fibroin water solution for preparing is nontoxic to human body, can be widely used in biomedical materials field, the needs that satisfied biomaterial development of new generation and used.
Embodiment
Below in conjunction with embodiment the present invention is further described:
Embodiment 1:
Prepare a kind of silk fibroin water solution with anti-gel property, step is as follows:
(1) silk comes unstuck
With the natural silk mass concentration 0.5% yellow soda ash (Na
2CO
3) solution (weight ratio of fibroin and distilled water is 1:20) boils 30min, clean up with clear water again; After so repeating not have soapy feeling to gained silk five times, it is cleaned, be placed in the vacuum drying oven 60 ℃ of fibroins after promptly obtaining coming unstuck after the oven dry down with distilled water.
(2) dissolving of fibroin
Be 1:8:2 preparation CaC1 in molar ratio
2/ H
2O/C
2H
5The OH ternary solvent, and filter, required clarification ternary solvent obtained; Fibroin after will coming unstuck was immersed in a certain amount of ternary solvent swelling three days, and constant temperature stirs and it fully dissolved in 4 hours in 50 ℃ of water-baths then.
(3) preparation of silk fibroin water solution
Fibroin after the dissolving dialyse in distilled water obtains silk fibroin water solution behind 3 days, centrifuging, places under 50 ℃ of the vacuum drying ovens to make it concentrated, makes massfraction and be 20% silk fibroin water solution.
(4) preparation of silk fibroin/vitamins C aqueous solution
In the 10g mass concentration is 20% silk fibroin water solution, add 0.01g, 0.02g, 0.04g vitamins C respectively, stirred 2 hours, make the silk fibroin/vitamins C aqueous solution, the i.e. anti-gelation silk fibroin water solution of different mixture ratio at 25 ℃ of lower magnetic forces.
To carry out gel time by the anti-gelation silk fibroin water solution of present embodiment technical scheme preparation measures, its method is: with mass concentration is that 20% the silk fibroin water solution and the silk fibroin/vitamins C aqueous solution of gained different mixture ratio place test tube to seal, leave standstill in 30 ℃ of thermostat water baths, survey its gel time.Other gets 10g concentration is that 20% silk fibroin water solution is sealed in vitro, places 4 ℃ of refrigerators then, observes its gel time, and as a comparison, the result is referring to table one.
Table 1 is that mass concentration is the gel time of the silk fibroin/vitamins C determination of aqueous solution of 20% silk fibroin water solution and different mixture ratio, and sample quality is 10g.Wherein silk fibroin/vitamins C aqueous solution is called blend solution, and the blend ratio is silk fibroin and ascorbic mass ratio.
Table 1
| Sample | Silk fibroin water solution (30 ℃) | Silk fibroin water solution (4 ℃) | Blend solution (200:1) | Blend solution (100:1) | Blend solution (50:1) |
| Gel time (my god) | 5 | 22 | 9 | 16 | 27 |
By table 1 as seen, 30 ℃ of following pure silk fibroin aqueous solution are placed and gel were promptly taken place in 5 days, and silk fibroin/vitamins C aqueous gel required time is obviously greater than the pure silk fibroin aqueous solution, and Vitamin C content is high more within the specific limits, and required time is long more.
Embodiment 2
Come unstuck and the dissolving step of fibroin with embodiment 1.
The preparation of silk fibroin water solution:
Fibroin after the dissolving dialyse in distilled water obtains silk fibroin water solution behind 3 days, centrifuging, places under 50 ℃ of the vacuum drying ovens to make it concentrated, makes massfraction respectively and be 30% silk fibroin water solution.
The preparation of silk fibroin/vitamins C blend aqueous solution:
Add 0.05g, 0.10g, 0.15g vitamins C respectively in the 10g mass concentration is 30% silk fibroin water solution, 25 ℃ of lower magnetic forces stirred 2 hours, made the silk fibroin/vitamins C aqueous solution of different mixture ratio.
To carry out gel time by the silk fibroin/vitamins C aqueous solution of present embodiment technical scheme preparation and measure, its method is with embodiment 1.
Mass concentration is that 30% the silk fibroin water solution and the silk fibroin/vitamins C aqueous gel time ratio of different mixture ratio see Table 2.Wherein silk fibroin/vitamins C aqueous solution is called blend solution, and the blend ratio is silk fibroin and ascorbic mass ratio.
Table 2
| Sample | Silk fibroin water solution (30 ℃) | Silk fibroin water solution (4 ℃) | Blend solution (60:1) | Blend solution (30:1) | Blend solution (20:1) |
| Gel time (my god) | 2 | 17 | 10 | 23 | 25 |
Can see equally by table 2, gel promptly took place in 2 days 30 ℃ of following pure silk fibroin aqueous solution placements, and silk fibroin/vitamins C aqueous gel required time is obviously greater than the plain aqueous solution of pure silk, and Vitamin C content is high more within the specific limits, and the gel required time is long more.
Claims (3)
1. anti-gelation silk fibroin water solution, its characteristics are: contain silk fibroin and antithrombotics vitamins C in the aqueous solution, wherein, the mass concentration of silk fibroin water solution is 20%～30%, and the ascorbic mass ratio of silk fibroin and antithrombotics is 200: 1～20: 1.
2. the preparation method of an anti-gelation silk fibroin water solution is characterized in that adopting following steps:
A. prepare silk fibroin quality concentration and be 20%～30% silk fibroin water solution;
B. add the antithrombotics vitamins C in the above-mentioned aqueous solution, the ascorbic mass ratio of silk fibroin and antithrombotics is 200: 1～20: 1, and constant temperature stirs in the water-bath below 50 ℃, obtains anti-gelation silk fibroin water solution.
3. the preparation method of a kind of anti-gelation silk fibroin water solution according to claim 2, its characteristics are: described silk fibroin water solution by natural silk through coming unstuck, obtaining after swelling, dissolving, dialysis, centrifugation and the concentration.
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| CN108852864A (en) * | 2018-07-04 | 2018-11-23 | 齐鲁工业大学 | A kind of graphene-silk-fibroin composite material and preparation method and application |
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