WO2022028107A1 - Procédé de préparation d'une solution composite de fibroine de soie et de séricine - Google Patents

Procédé de préparation d'une solution composite de fibroine de soie et de séricine Download PDF

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Publication number
WO2022028107A1
WO2022028107A1 PCT/CN2021/099614 CN2021099614W WO2022028107A1 WO 2022028107 A1 WO2022028107 A1 WO 2022028107A1 CN 2021099614 W CN2021099614 W CN 2021099614W WO 2022028107 A1 WO2022028107 A1 WO 2022028107A1
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silk
sericin
silk fibroin
solution
water
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Chinese (zh)
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王建南
刘苹
裔洪根
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Suzhou University
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Suzhou University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms

Definitions

  • the invention relates to the technical field of biomedical materials, in particular to a preparation method of a silk fibroin sericin composite solution.
  • tissue repair methods include traditional autologous transplantation, allogeneic transplantation and artificial tissue engineering scaffold transplantation.
  • autologous transplantation repair has the best therapeutic effect, it has serious constraints on many factors such as damage to the donor site, insufficient dose and complications; allogeneic transplantation faces problems such as immune rejection and disease transmission; tissue engineering was proposed in the 1990s. , and then the research on tissue engineering scaffold materials has been rapidly developed. Synthetic polymer materials in tissue engineering have the advantages of wide sources, tunable physicochemical properties, and excellent processability.
  • tissue engineering scaffolds Lack of biological activity, poor cytocompatibility, no degradation or degradation products affecting the tissue microenvironment. In situ induced tissue regeneration is considered to be the most ideal mode for repairing defective or diseased tissue, so natural polymers are widely favored in the research of tissue engineering scaffolds due to their excellent bioactivity and degradability, such as collagen, gelatin, and alginate. , hyaluronic acid, chitosan, etc.
  • the mechanical properties of most materials are very poor, and the application research as engineering scaffolds for tissues (such as bones, teeth, etc.) that require high mechanical properties still faces great challenges.
  • Silk fibroin and sericin have excellent cytocompatibility, hemocompatibility and histocompatibility, and controllable molding.
  • tissue engineering scaffold materials they have been widely used in tissue engineering applications such as skin, blood vessels, and bones.
  • the currently studied regenerated silk (silk fibroin, sericin) protein scaffolds also have poor mechanical properties and are difficult to meet the requirements for replacement of hard tissues such as bones and teeth.
  • the macromolecular structure of vellum and sericin is greatly damaged, and the degree of polymerization (molecular weight) of the polymer is an extremely important intrinsic factor that determines the performance of the polymer.
  • the protein in silk has high purity, unique composition, and the preparation method can be relatively simple and mild, that is, it can control the damage to silk macromolecules, unlike collagen. Purification is complicated.
  • the silk fibroin in the inner core of silk accounts for 70-80% of the silk mass.
  • the macromolecules are connected by disulfide bonds with six heavy chains of about 390 kDa and six light chains of about 26 kDa, and then hydrophobic with another glycoprotein/P25 of about 25 kDa.
  • the heavy chain has the basis of the sequence structure to form high-performance materials; the sericin covering the silk accounts for 20-30% of the silk quality, and the molecular weight of sericin is also relatively large, and there are as many as 9 kinds. From the histological point of view, it is pointed out that there are three layers of sericin, namely, sericin, sericin, and sericin. And the internal solubility is getting lower and lower, and the ⁇ conformation increases.
  • the regenerated silk fibroin solutions are all obtained by dissolving strong alkali weak acid salts or enzyme degumming salts. These methods cause the destruction of silk fibroin macromolecules; the regenerated sericin solutions are mainly derived from silkworm cocoons, and the two solutions are generally prepared independently.
  • CN02138129.1 discloses the preparation of a silk fibroin and sericin composite tissue engineering scaffold. The composite material is prepared by mixing pre-prepared regenerated silk fibroin and regenerated sericin in proportion.
  • the degumming method uses a strong base and weak acid salt solvent, and sericin is extracted from the outer part of the silkworm cocoon silk covered with sericin.
  • CN201510353363.8 discloses a bone tissue engineering scaffold material containing silk fibroin and sericin and a preparation method thereof.
  • the regenerated silk fibroin and regenerated sericin are also obtained in steps and then mixed to prepare the scaffold material.
  • the preparation of silk fibroin does not disclose specific process conditions.
  • the sericin is obtained from silk cocoons and treated with organic solvents such as alcohol.
  • the technical problem solved by the present invention is to provide a preparation method of a silk fibroin sericin composite solution, which has the characteristics of high viscosity and high molecular weight.
  • the present application provides a preparation method of a silk fibroin sericin composite solution, comprising the following steps:
  • A) silk is placed in warm water for pretreatment, then placed in 65 ⁇ 95 °C of water for treatment, and dried to obtain the silk of initial treatment;
  • the temperature of the warm water is 35 ⁇ 60° C., and the time of the pretreatment is 1 ⁇ 10 min.
  • the method further includes: taking out the pretreated silk, washing and dehydrating in water at 25-30°C.
  • the treatment time in water at 65-95° C. is 1-8 h.
  • the ratio of the silk to warm water is 1 g: (40-60) ml
  • the ratio of the pretreated silk to water is 1 g: (40-60) ml.
  • the method before the drying after the treatment, further comprises: washing the treated silk in water at 25-30°C and then dehydrating; the drying adopts hot air drying at 40-60°C.
  • the neutral salt solution is an 8-10 M lithium bromide aqueous solution, and the ratio of the initially treated silk to the lithium bromide aqueous solution is 1 g: (20-30) ml.
  • the temperature of the treatment is 50-80°C.
  • the purifying method is specifically:
  • the obtained silk fibroin sericin composite solution is poured into a dialysis bag with a molecular weight cut-off of 10-50 kDa, and placed in deionized water for continuous dialysis for 1-3 days.
  • the present application provides a method for preparing a silk fibroin sericin composite solution, which comprises first placing the silk in warm water for pretreatment, and then placing it in water for treatment to obtain initially treated silk; and then treating it in a neutral salt solution , the silk fibroin sericin composite solution is obtained after purification; the present application adopts the mild degumming and one-step dissolution method to prepare the silk fibroin sericin composite solution.
  • the pretreatment in warm water and the subsequent treatment in water at a specific temperature make the silk surface
  • the sericin is partially or completely removed, while the silk fibroin macromolecular chain will not be destroyed in the subsequent degumming process during the whole process of the special warm water treatment.
  • the present application uses deionized water, mild degumming, and one-step dissolution to prepare large-molecular-weight and high-viscosity silk fibroin sericin.
  • the method can avoid the damage of the silk fibroin fiber macromolecules, and finally the silk fibroin and sericin in the prepared silk fibroin and sericin composite solution have high viscosity and large molecular weight.
  • the embodiment of the present invention discloses a preparation method of a silk fibroin sericin composite solution, which includes the following steps:
  • A) silk is placed in warm water for pretreatment, then placed in 65 ⁇ 95 °C of water for treatment, and dried to obtain the silk of initial treatment;
  • the silk is placed in warm water for pretreatment, and then placed in water at 65-95° C. for treatment, and then dried to obtain the pre-treated silk; in this process, The sericin of silk is completely or partially dissolved, while the silk fibroin is still in the state of fiber. In water, it is in solution state.
  • the temperature of the warm water is 35-60°C, that is, the silk can be pretreated in warm water at 35°C, 40°C, 45°C, 50°C, 55°C or 60°C, and the ratio of the silk to the warm water is 1 g : (40-60) ml, more specifically, the ratio of the silk to the warm water is 1g: 42ml, 1g: 45ml, 1g: 48ml, 1g: 52ml, 1g: 55ml, 1g: 58ml.
  • the pretreatment is carried out in the above-mentioned warm water at a constant temperature, and the time of the pretreatment is 1-10 minutes, and the above-mentioned pretreatment is always carried out in stirring, so as to ensure that the silk can be initially and completely submerged in the water.
  • the pretreated silk is taken out and washed and dehydrated in deionized water at 25-30°C.
  • the dehydrated silk is treated in water at 65 to 95°C. , 90 °C or 93 °C water treatment.
  • the method that the silk is pretreated in water and then treated in water does not involve any reagent treatment, and can dissolve sericin while protecting the macromolecular structure of silk fibroin from being destroyed.
  • the ratio of the pretreated silk to water is 1g:(40 ⁇ 60)ml, more specifically, the ratio of the pretreated silk to water is 1g:42ml, 1g:45ml, 1g:48ml, 1g : 50ml, 1g: 52ml, 1g: 55ml, 1g: 58ml or 1g: 60ml.
  • the degumming rate of silk sericin can be adjusted according to the different temperature and time of the water, so as to realize the preparation of the composite solution of silk fibroin sericin with different viscosities and different molecular weights; in this application, the water treatment time is preferably 1-8h , so that the silk is partially or completely degummed; more specifically, the water treatment time is 1.5h, 2h, 2.5h, 3h, 3.2h, 3.6h, 4.2h, 4.5h, 5.0h, 5.5h, 6.0h , 6.5h, 6.8h, 7.0h or 7.5h. After the treatment, the treated silk is washed in water at 25-30°C, then dehydrated and dried; the drying adopts hot air drying at 40-60°C.
  • the silk undergoes the above-mentioned treatment to obtain the initially treated silk, which is still in a fibrous state and realizes the protection of silk fibroin, so as to ensure the high molecular weight and high viscosity of silk fibroin sericin.
  • the initially treated silk is then treated in a neutral salt solution and purified to obtain a silk fibroin sericin composite solution.
  • the fibrous silk that is initially treated is dissolved to obtain a composite solution of silk fibroin sericin.
  • the inner core of the silk is silk fibroin, and the periphery is wrapped with different contents of sericin.
  • the neutral salt solution is specifically selected from the lithium bromide aqueous solution in this application, and its concentration is 8-10M, and the ratio of the initially treated silk to the lithium bromide aqueous solution is 1 g: (20-30) ml, more specifically, The ratio of the initially treated silk to the lithium bromide aqueous solution is 1g:22ml, 1g:24ml, 1g:25ml, 1g:27ml or 1g:29ml.
  • the temperature of the treatment is specifically 50-80°C, and more specifically, the temperature of the treatment is specifically 52°C, 57°C, 62°C, 65°C, 67°C, 68°C, 72°C, 75°C or 78°C.
  • the obtained silk fibroin sericin dissolving solution is purified to remove lithium bromide and low molecular weight silk fibroin sericin; the purification method is specifically pouring the obtained silk fibroin sericin complex dissolving solution into Place in a dialysis bag with a molecular weight cut-off of 10-50kDa and place it in deionized water for continuous dialysis for 1-3 days.
  • the invention adopts the mild degumming and one-step dissolving method to prepare the silk fibroin and sericin composite solution, which breaks through the traditional separate preparation of silk fibroin and sericin, which are respectively dissolved and mixed before being used to prepare the composite material. It is important that the molecular weight of silk protein in the composite solution is concentrated above 150kDa, or contains a molecular weight of about 25kDa, while the molecular weight of the silk fibroin solution prepared by the reported conventional method or the solution of silk fibroin sericin dissolved and mixed shows the molecular weight. Continuous distribution, mainly distributed below 85kDa to 15kDa or even smaller.
  • the silk is treated with hot water, so that the silk fibroin macromolecular chain (especially the heavy chain) will not be destroyed during the degumming process; From the outside to the inside of the coating layer, the types, structures and properties of sericin are also different.
  • silk fibroin sericin composite solutions with different compositions and materials with different properties (especially mechanical properties) are obtained. .
  • the viscosity of the silk protein solution of the one-step dissolution method is much higher than that of the conventionally reported mixed solution.
  • the present invention provides a new technology for the preparation of high-performance regenerated silk protein material based on the molecular weight of the polymer, which is the fundamental factor affecting the mechanical properties of the polymer, and will solve the problem of natural biopolymer tissue that can induce regeneration and repair of hard tissue.
  • the degumming rate of the silk treated in steps 1 and 2 was measured by weighing method, and the degumming rate was measured to be 8%.
  • the dried silk is weighed and dissolved in a 9.3M lithium bromide aqueous solution at a bath ratio of 1:25 (g/mL), and treated at 65° C. until the silk is completely dissolved to obtain a Bombyx mori silk fibroin sericin composite solution;
  • the viscosity of sericin (11Pa.s and 40Pa.s) is much higher than that of silk fibroin, and the presence of sericin will increase the viscosity of the solution, so the extreme value of the viscosity of the solution in this example under shearing action is lower than that of Example 1.
  • the degumming rate of the silk treated in steps 1 and 2 was measured by weighing method, and the degumming rate was measured to be 17%.
  • the dried silk is weighed and dissolved in a 9.3M lithium bromide aqueous solution at a bath ratio of 1:25 (g/mL), and treated at 65° C. until the silk is completely dissolved to obtain a Bombyx mori silk fibroin sericin composite solution;
  • the degumming rate of the silk treated in steps 1 and 2 was measured by weighing method, and the degumming rate was measured to be 17%.
  • dehydrated silk was placed in deionized water preheated at 90°C and boiled at a constant temperature for 7 hours according to a liquor ratio of 1:50 (g/mL), and the silk was taken out and fully cleaned with deionization at 25-30°C, and dehydrated. Then dry it with hot air at 40-60°C;
  • the dried silk is weighed and dissolved in a 9.3M lithium bromide aqueous solution at a bath ratio of 1:25 (g/mL), and treated at 65° C. until the silk is completely dissolved to obtain a Bombyx mori silk fibroin sericin composite solution;
  • the silk fibroin sericin composite solution was poured into a dialysis bag with a molecular weight cut-off of 14kDa, and the wall of the dialysis bag was a semi-permeable membrane. Place the dialysis bag filled with silk fibroin sericin solution in a container filled with deionized water, replace the water in the container with new deionized water every 2 hours, and continue dialysis for 3 days to obtain a purified home.
  • Silk fibroin sericin complex aqueous solution placed into a dialysis bag with a molecular weight cut-off of 14kDa, and the wall of the dialysis bag was a semi-permeable membrane.
  • the degumming rate of the silk treated in steps 1 and 2 was measured by weighing method, and the degumming rate was measured to be 23%.
  • the degumming rate is the same, that is, the sericin is completely removed, and the dialysis molecular weight is the same.
  • the molecular weight of the silk solution degummed by hot water in this example is not only large in molecular weight but also in a concentrated distribution.
  • Conventional sodium carbonate degumming The molecular weight of the silk solution is small and the distribution is very scattered; the larger the molecular weight, the higher the viscosity, and the smaller the molecular weight polydispersity coefficient, the higher the viscosity. times and 12.5 times.
  • the dried silk is weighed and dissolved in a 9.3M lithium bromide aqueous solution at a bath ratio of 1:25 (g/mL), and treated at 65° C. until the silk is completely dissolved to obtain a Bombyx mori silk fibroin sericin composite solution;
  • the degumming rate of the silk treated in the first and second steps is measured by the weighing method, and the degumming rate is measured to be 23%.
  • the viscosity value and the viscosity extreme value under the initial shear action measured in this embodiment are Comparative Example 1 40 times and 21 times.
  • the Bombyx mori fibroin dissolving solution is perfused in a dialysis bag with a molecular weight cut-off of 14kDa, the wall of the dialysis bag is a semi-permeable membrane, and the dialysis bag perfused with the Bombyx mori silk fibroin dissolving solution is placed in a container filled with deionized water, The water in the container was replaced with new deionized water every 2 hours, and the dialysis continued for 3 days to obtain the purified silk fibroin aqueous solution;
  • a rotary evaporator to concentrate or dilute silk fibroin with deionized water to obtain an aqueous solution of a certain mass fraction.
  • the molecular weight distribution of the silk fibroin solution is measured by polyacrylamide gel electrophoresis, and it is mainly distributed below 85kDa, although the same A dialysis bag with a molecular weight cut-off of 14 kDa was used, but the content of silk fibroin below 15 kDa was still high.
  • the degumming rate of the silk treated in the first and second steps was measured by the weighing method, and the degumming rate was measured to be 23%.
  • the Bombyx mori fibroin dissolving solution is perfused in a dialysis bag with a molecular weight cut-off of 14kDa, the wall of the dialysis bag is a semi-permeable membrane, and the dialysis bag perfused with the Bombyx mori silk fibroin dissolving solution is placed in a container filled with deionized water, The water in the container was replaced with new deionized water every 2 hours, and the dialysis continued for 3 days to obtain the purified silk fibroin aqueous solution;
  • the dehydrated silk is placed in preheated deionized water at 90°C and cooked at a constant temperature for 7 hours according to the liquor ratio of 1:50 (g/mL), the fibers are discarded, and the sericin aqueous solution is collected and freeze-dried to obtain solid sericin;
  • the molecular weight and viscosity of the silk fibroin-sericin composite aqueous solutions prepared in Examples 1-5 are significantly higher than those of the silk protein solutions obtained by conventional degumming methods in Comparative Examples 1-2.

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Abstract

L'invention concerne un procédé de préparation d'une solution composite de fibroïne de soie et de séricine, le procédé comprenant les étapes suivantes : A) placer la soie dans de l'eau chaude pour le prétraitement, puis la placer dans de l'eau à 65-95°C pour le traitement, et la sécher afin d'obtenir la soie initialement traitée ; et B) traiter la soie initialement traitée dans une solution de sel neutre, puis purifier la solution de dissolution composite de fibroïne et de séricine obtenue pour obtenir la solution composite de fibroïne et de séricine. Selon la présente invention, la solution composite de fibroïne de soie et de séricine est préparée en utilisant un procédé de dégommage doux et de dissolution en une étape, qui surpasse le procédé traditionnel selon lequel la fibroïne de soie et la séricine sont préparées séparément, dissoutes séparément et ensuite mélangées pour une utilisation dans la préparation d'un matériau composite. Plus important, le poids moléculaire de la protéine de soie dans la solution composite est concentré à 150 kDa ou plus. Toutefois, le poids moléculaire rapporté d'une solution de fibroïne de soie ou d'une solution dans laquelle la fibroïne de soie et la séricine sont dissoutes, puis mélangées qui est préparé en utilisant un procédé conventionnel montre une distribution continue, étant principalement distribuée à 85 kDa ou moins à 15 kDa ou même moins.
PCT/CN2021/099614 2020-08-06 2021-06-11 Procédé de préparation d'une solution composite de fibroine de soie et de séricine Ceased WO2022028107A1 (fr)

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CN114790300A (zh) * 2022-04-29 2022-07-26 中国科学院青岛生物能源与过程研究所 一种丝素蛋白纳米粒的制备方法
CN115247198A (zh) * 2022-06-14 2022-10-28 上海中匡生物科技有限公司 一种制备丝素多肽的方法及其应用
CN119736791A (zh) * 2025-03-04 2025-04-01 杭州万事利丝绸数码印花有限公司 一种蚕丝保胶工艺及高丝胶蚕丝

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CN115531284A (zh) * 2022-09-15 2022-12-30 江苏华佳丝绸股份有限公司 一种含丝胶蛋白的蚕丝面膜的制备方法
CN119264477B (zh) * 2024-09-24 2026-04-24 苏州大学 一种可见光固化丝素/丝胶蛋白水凝胶的制备方法

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