WO2020259672A1 - 一种促进凝血的丝素-多肽电纺膜及其制备方法 - Google Patents

一种促进凝血的丝素-多肽电纺膜及其制备方法 Download PDF

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WO2020259672A1
WO2020259672A1 PCT/CN2020/098423 CN2020098423W WO2020259672A1 WO 2020259672 A1 WO2020259672 A1 WO 2020259672A1 CN 2020098423 W CN2020098423 W CN 2020098423W WO 2020259672 A1 WO2020259672 A1 WO 2020259672A1
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polypeptide
silk fibroin
electrospun membrane
blood coagulation
fibroin
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杨明英
雷芳
帅亚俊
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浙江大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A61L24/0015—Medicaments; Biocides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/0005—Ingredients of undetermined constitution or reaction products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/043—Mixtures of macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/10—Polypeptides; Proteins
    • A61L24/108—Specific proteins or polypeptides not covered by groups A61L24/102 - A61L24/106
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • 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
    • A61L2300/418—Agents promoting blood coagulation, blood-clotting agents, embolising agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/04—Materials for stopping bleeding

Definitions

  • the invention relates to the field of blood coagulation materials, in particular to a silk fibroin-polypeptide electrospun membrane for promoting blood coagulation and a preparation method thereof.
  • coagulation materials The research and application of coagulation materials has always been an important field of medical research. Whether it is traumatic bleeding caused by daily life, clinical surgery or even war, the application of blood coagulation materials is of great significance for rapid hemostasis and life saving.
  • the essence of blood coagulation is the process by which fibrinogen in the blood becomes fibrin.
  • Fibrinogen as coagulation factor I, plays a huge role in the coagulation function. Therefore, it has a coagulation material that can target fibrinogen. It can be highly enriched in human fibrinogen, which is conducive to the formation of fibrin network structure at the wound site and further promotes blood coagulation.
  • GPRPPSEHLQIT SED ID NO: 1
  • GPRPPSEHLQIT SED ID NO: 1
  • the peptides Compared with materials that display this sequence, such as bacteriophages, the peptides have higher purity, which can enrich human fibrinogen to a greater extent on the surface of the material, and the peptides have better safety and biocompatibility, making them more suitable for use biomaterials.
  • the electrospun membrane prepared by the electrospinning technology has a nanofiber structure, and has a larger specific surface area and greater porosity. Its structural characteristics are similar to many natural tissues and organs.
  • the electrospun membrane made of silk fibroin is often used Make biological materials.
  • the rough surface structure of silk fibroin electrospun membrane has better adhesion to protein under the blood flow state than other smooth surface coagulation materials, which is conducive to the formation of thrombus in the wound, promotes blood coagulation and wound healing.
  • traditional silk fibroin electrospun membranes can only adhere to proteins through the effects of electrostatic adsorption on the surface, and cannot target binding proteins.
  • Human fibrinogen as a protein that can significantly affect the coagulation ability, in order to target binding on the surface of the material
  • we added the polypeptide GPRPPSEHLQIT SED ID NO:1 that can target and bind to human fibrinogen, and made a silk fibroin-polypeptide electrospun membrane.
  • the electrospun membrane can target human fibrinogen in the blood on the surface, which is beneficial to the formation of a fibrin network structure on the surface of the material and promotes wound coagulation.
  • the present invention provides a silk fibroin-polypeptide electrospun membrane capable of targeted binding to human fibrinogen and a preparation method thereof.
  • the present invention is an electrospun membrane made of silk fibroin as a carrier and added with polypeptide GPRPPSEHLQIT (SED ID NO: 1), which has better coagulation effect than traditional blood coagulation materials and pure silk fibroin electrospun membrane.
  • the silk fibroin-polypeptide electrospun membrane prepared by the invention is mainly used to promote blood coagulation, and is a blood coagulation material that can target and bind human fibrinogen.
  • the specific technical scheme of the present invention is as follows:
  • the invention discloses a silk fibroin-polypeptide electrospun membrane that promotes blood coagulation.
  • the used polypeptide sequence is GPRPPSEHLQIT (SED ID NO: 1), and the polypeptide concentration is 0.2 mg/mL-10 mg/mL.
  • the electrospun membrane of the present invention is interwoven with silk fibroin nanofibers, and polypeptides are evenly distributed in the nanofibers.
  • the invention also discloses a preparation method of a silk fibroin-polypeptide electrospun membrane that promotes blood coagulation, and the specific preparation steps adopted are as follows:
  • step 2) Electrospinning the mixed solution obtained in step 2) to obtain a silk fibroin-polypeptide electrospun membrane.
  • the mass ratio of the lyophilized silk fibroin powder: hexafluoroisopropanol solution used in step 2) of the present invention is 2:98-20:80.
  • the concentration of the polypeptide GPRPPSEHLQIT (SED ID NO: 1) added in step 2) of the present invention is 0.2 mg/mL-10 mg/mL.
  • the silk fibroin-polypeptide electrospun membrane prepared in step 3) of the present invention is treated with alcohol to obtain a water-insoluble silk fibroin-polypeptide electrospun membrane.
  • the present invention has the following outstanding features:
  • the polypeptide used in the present invention is a polypeptide obtained by self-screening. Compared with other polypeptides, it can specifically target and bind to human fibrinogen.
  • the polypeptide enriches human fibrinogen on the electrospun membrane, which is beneficial to the membrane Form a fibrin network structure on the surface to promote blood clotting;
  • the polypeptide used in the electrospun membrane has higher biological safety than the phage expressing the polypeptide sequence
  • the silk fibroin used in the electrospun membrane is a natural polymer protein. Compared with other inorganic materials or synthetic polymer materials, it has better biocompatibility, higher safety, and is convenient to manufacture, which is convenient for mass production.
  • Figure 1 is an electron micrograph of a silk fibroin-polypeptide electrospun membrane.
  • Fig. 2 is a graph showing the activated partial thromboplastin time (APTT) results of silk fibroin-polypeptide electrospun membranes with different quality peptides (P) and pure silk fibroin electrospun membranes.
  • APTT activated partial thromboplastin time
  • Figure 3 is a graph showing the results of the coagulation index of electrospun membranes with added polypeptide GPRPPSEHLQIT (SED ID NO: 1) and different sequence polypeptides.
  • step 2 Electrospin the solution obtained in step 2) to obtain a silk fibroin-polypeptide electrospun membrane. Observe the electrospun membrane with a scanning electron microscope to obtain Figure 1.

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Abstract

本发明公开了一种促进凝血的丝素-多肽电纺膜及其制备方法，以家蚕丝素蛋白为载体，添加多肽GPRPPSEHLQIT制成的电纺膜，主要用于促进凝血，是一种可以靶向结合人纤维蛋白原的凝血材料，包括将蚕茧脱胶后溶解、过滤、透析、浓缩、冻干，获得丝素冻干粉等步骤，本发明中使用的多肽为自行筛选得到的多肽，对比其余多肽，可以特异性靶向结合人纤维蛋白原，该多肽通过在电纺膜上富集人纤维蛋白原，有利于在膜表面形成纤维蛋白网状结构，促进凝血，该电纺膜所使用的丝素为天然高分子蛋白，对比其余无机材料或人工合成高分子材料，生物相容性更好，安全性更高，且制作方便，便于量产。

Description

一种促进凝血的丝素-多肽电纺膜及其制备方法 技术领域
本发明涉及凝血材料领域，具体地说，是一种促进凝血的丝素-多肽电纺膜及其制备方法。
背景技术
凝血材料的研究与应用一直是医学研究的重要领域。无论是日常生活，临床手术甚至战争等造成的创伤流血，促凝血材料的应用对于快速止血、挽救生命等具有重要意义。而血液凝固的本质便是血液中纤维蛋白原变成纤维蛋白的过程，纤维蛋白原作为凝血因子I，对凝血功能起到了巨大的作用，因此，具有能够靶向结合纤维蛋白原能力的凝血材料可以高度富集人纤维蛋白原，有利于创伤部位纤维蛋白网状结构的形成，进一步促进凝血。
家蚕丝素蛋白作为一种天然高分子蛋白，具有极好的生物相容性、可降解性、易于加工成型和良好的力学性能等优点，常被加工成为膜、凝胶、支架等材料用于再生医学等领域。多肽GPRPPSEHLQIT(SED ID NO：1)能够靶向结合人纤维蛋白原，加入凝血材料中可以使人纤维蛋白原富集在材料表面，进而促进凝血。相对于噬菌体等展示该段序列的材料，多肽的纯度更高，能够更大程度地在材料表面富集人纤维蛋白原，而且多肽的安全性、生物相容性都更优秀，更适合用于生物材料。静电纺丝技术制备的电纺膜具有纳米纤维结构，且具有更大的比表面积以及更大的孔隙率，其结构特征与许多天然组织器官相似，由丝素制成的电纺膜常被用做生物材料。丝素电纺膜的粗糙表面结构对比其余表面光滑的凝血材料，在血液流动状态下对蛋白有更好的黏附力，利于在伤口处形成血栓，促进血液凝固，伤口愈合。但传统的丝素电纺膜只能通过表面的静电吸附等作用黏附蛋白，不能靶向地结合蛋白，而人纤维蛋白原作为能显著影响凝血能力的蛋白，为了能在材料表面靶向地结合人纤维蛋白原，我们添加了能够靶向结合人纤维蛋白原的多肽GPRPPSEHLQIT(SED ID NO：1)，制成了丝素-多肽电纺膜。该电纺膜可以在表面靶向地结合血液中的人纤维蛋白原，有利于在材料表面形成纤维 蛋白网络状结构，促进伤口凝血。
发明内容
本发明针对传统凝血材料领域的不足，提供了一种可以靶向结合人纤维蛋白原的丝素-多肽电纺膜及其制备方法。本发明为以家蚕丝素蛋白为载体，添加多肽GPRPPSEHLQIT(SED ID NO：1)制成的电纺膜，对比传统的凝血材料以及纯丝素电纺膜的凝血效果要更佳。本发明制备的丝素-多肽电纺膜主要用于促进凝血，是一种可以靶向结合人纤维蛋白原的凝血材料。本发明的具体技术方案如下：
本发明公开了一种促进凝血的丝素-多肽电纺膜，所使用的多肽序列为GPRPPSEHLQIT(SED ID NO：1)，多肽浓度为0.2mg/mL——10mg/mL。
作为进一步地改进，本发明所述的电纺膜为丝素纳米纤维交织而成，多肽均匀分布在纳米纤维中。
本发明还公开了一种促进凝血的丝素-多肽电纺膜的制备方法，所采用的具体制备步骤如下：
1)将蚕茧脱胶后溶解、过滤、透析、浓缩、冻干，获得丝素冻干粉；
2)将丝素冻干粉、多肽GPRPPSEHLQIT(SED ID NO：1)与六氟异丙醇溶液混合均匀；
3)将步骤2)所得混合溶液进行静电纺丝，即得到丝素-多肽电纺膜。
作为进一步地改进，本发明所述的步骤2)使用的丝素冻干粉：六氟异丙醇溶液的质量比为2：98——20：80。
作为进一步地改进，本发明所述的步骤2)添加的多肽GPRPPSEHLQIT(SED ID NO：1)浓度为0.2mg/mL——10mg/mL。
作为进一步地改进，本发明所述的步骤3)制备所得丝素-多肽电纺膜经过醇类处理，得到不溶于水的丝素-多肽电纺膜。
与现有技术相比，本发明具有以下突出特点：
(1)本发明中使用的多肽为自行筛选得到的多肽，对比其余多肽，可以特异性靶向结合人纤维蛋白原，该多肽通过在电纺膜上富集人纤维蛋白原，有利于在膜表面形成纤维蛋白网状结构，促进凝血；
(2)该电纺膜所使用的多肽，与表达该段多肽序列的噬菌体相比，生物安全性更高；
(3)该电纺膜所使用的丝素为天然高分子蛋白，对比其余无机材料或人工合成高分子材料，生物相容性更好，安全性更高，且制作方便，便于量产。
附图说明
图1是丝素-多肽电纺膜的电镜图。
图2是添加不同质量多肽(P，peptide)的丝素-多肽电纺膜与单纯的丝素电纺膜的活化部分凝血活酶时间(APTT，activated partial thromboplastin time)结果图。
图3是添加多肽GPRPPSEHLQIT(SED ID NO：1)与添加不同序列多肽的电纺膜的凝血指数结果图。
具体实施方式
下面通过具体实施例对本发明的技术方案作进一步的详细说明，以下实施例是对本发明的解释而本发明并不局限于以下实施例。
实施例1
(1)将蚕茧脱胶后溶解、过滤、透析、浓缩得到丝素溶液，再经冻干获得丝素冻干粉；
(2)将2g丝素冻干粉与8g六氟异丙醇溶液混合，取1mL该混合液加入10mg多肽GPRPPSEHLQIT(SED ID NO：1)，得到步骤2)所得溶液；
(3)将步骤2)所得溶液进行静电纺丝，即得到丝素-多肽电纺膜，将该电纺膜进行扫描电镜的观察，可得到图1。
实施例2
(1)将蚕茧脱胶后溶解、过滤、透析、浓缩得到丝素溶液，再经冻干获得丝素冻干粉；
(2)将1g丝素冻干粉与13.29g六氟异丙醇溶液混合，分别取1mL该混合液加入0、0.2、0.4、0.6、1mg多肽GPRPPSEHLQIT(SED ID NO：1)，得 到步骤2)所得溶液；
(3)将步骤2)所得溶液分别进行静电纺丝，即得到丝素-多肽电纺膜；
(4)将这些电纺膜进行活化部分凝血活酶时间(APTT，activated partial thromboplastin time)检测，可得到图2的结果，说明随着多肽浓度上升，所需时间会变短，表示多肽浓度越高，凝血效果越好。
实施例3
(1)将蚕茧脱胶后溶解、过滤、透析、浓缩得到丝素溶液，再经冻干获得丝素冻干粉；
(2)将0.2g丝素冻干粉与9.8g六氟异丙醇溶液混合，分别取1mL该混合液加入0.2mg多肽GPRPPSEHLQIT(SED ID NO：1)，另取1mL该混合液加入0.2mg别的多肽用于对照，得到步骤2)所得溶液；
(3)将步骤2)所得溶液分别进行静电纺丝，即得到丝素-多肽电纺膜；
(4)将上述电纺膜与新鲜血液混合分别在37℃孵育0.5、1、2、3、4min，取出凝血块后，检测未凝血液中的血红蛋白含量，占凝血前总血红蛋白含量的比例，为凝血指数BCI，可得图3，指数下降越快，表示凝血速度越快，说明加入多肽GPRPPSEHLQIT(SED ID NO：1)的电纺膜比加入别的多肽的电纺膜促凝血效果更好。
实施例4
(1)将蚕茧脱胶后溶解、过滤、透析、浓缩得到丝素溶液，再经冻干获得丝素冻干粉；
(2)将1g丝素冻干粉与9g六氟异丙醇溶液混合，取1mL该混合液加入5mg多肽GPRPPSEHLQIT(SED ID NO：1)，得到步骤2)所得溶液；
(3)将步骤2)所得溶液进行静电纺丝，即得到丝素-多肽电纺膜；
(4)将步骤3)得到的电纺膜浸泡在90％乙醇中30min，得到不溶于水的丝素-多肽电纺膜。
最后，还需要注意的是，以上列举的仅是本发明的具体实施例子。显然，本 发明不限于以上实施例子，还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。

Claims (6)

  1. 一种促进凝血的丝素-多肽电纺膜，其特征在于，所使用的多肽序列为GPRPPSEHLQIT(SED ID NO：1)，多肽浓度为0.2mg/mL——10mg/mL。
  2. 根据权利要求1所述丝素-多肽电纺膜，其特征在于，所述的电纺膜为丝素纳米纤维交织而成，多肽均匀分布在纳米纤维中。
  3. 一种如权利要求1或2所述的促进凝血的丝素-多肽电纺膜的制备方法，其特征在于，所采用的具体制备步骤如下：
    1)将蚕茧脱胶后溶解、过滤、透析、浓缩、冻干，获得丝素冻干粉；
    2)将丝素冻干粉、多肽GPRPPSEHLQIT(SED ID NO：1)与六氟异丙醇溶液混合均匀；
    3)将步骤2)所得混合溶液进行静电纺丝，即得到丝素-多肽电纺膜。
  4. 根据权利要求3所述的丝素-多肽电纺膜的制备方法，其特征在于，所述的步骤2)使用的丝素冻干粉：六氟异丙醇溶液的质量比为2：98——20：80。
  5. 根据权利要求3或4所述的丝素-多肽电纺膜的制备方法，其特征在于，所述的步骤2)添加的多肽GPRPPSEHLQIT(SED ID NO：1)浓度为0.2mg/mL——10mg/mL。
  6. 根据权利要求3所述的丝素-多肽电纺膜的制备方法，其特征在于，所述的步骤3)制备所得丝素-多肽电纺膜经过醇类处理，得到不溶于水的丝素-多肽电纺膜。
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