WO2023273875A1 - 一种丝胶粘合剂的制备方法及应用 - Google Patents

一种丝胶粘合剂的制备方法及应用 Download PDF

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WO2023273875A1
WO2023273875A1 PCT/CN2022/098798 CN2022098798W WO2023273875A1 WO 2023273875 A1 WO2023273875 A1 WO 2023273875A1 CN 2022098798 W CN2022098798 W CN 2022098798W WO 2023273875 A1 WO2023273875 A1 WO 2023273875A1
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sericin
adhesive
preparation
solution
reduced
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PCT/CN2022/098798
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English (en)
French (fr)
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王琳
王征
刘欢
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华中科技大学同济医学院附属协和医院
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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
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/10—Polypeptides; Proteins
    • A61L24/108—Specific proteins or polypeptides not covered by groups A61L24/102 - A61L24/106
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A61L24/0015—Medicaments; Biocides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A61L24/0031—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A61L24/0042—Materials resorbable by the body
    • 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
    • A61L24/02—Surgical adhesives or cements; Adhesives for colostomy devices containing inorganic 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/04—Materials for stopping bleeding

Definitions

  • the invention belongs to the field of medical biological composite materials, in particular to a preparation method and application of a sericin adhesive.
  • water molecules can form a thin hydration layer at the interface between the biomaterial and the tissue, preventing adhesion groups on the biomaterial from reaching the tissue surface.
  • water molecules can easily disrupt the non-covalent adhesive interactions between biomaterials and tissues.
  • swelling or shrinking of biomaterials (such as hydrogels) after absorbing water can also weaken their adhesion to tissues. Therefore, it is of great significance to develop biomaterials that can achieve effective adhesion under wet conditions.
  • Sericin is a natural macromolecular protein wrapped on the surface of silk fibres.
  • sericin has good biological properties such as low immunogenicity, biodegradability, anti-oxidation, cell adhesion, so sericin has a certain effect on skin tissue, vascular tissue, bone tissue damage. Excellent restoration effect.
  • the present invention provides the preparation method and application of the underwater sericin adhesive, which promotes the drainage of the tissue surface by adjusting the hydrophilic-hydrophobic balance of the adhesive components, and mainly solves the problem that the existing adhesive does not have the ability to bind underwater. Weak adhesive strength, poor biocompatibility and other issues.
  • the present invention adopts the following technical solutions:
  • the preparation method of sericin adhesive comprises the steps
  • Preparation of reduced sericin dry dissolving sericin powder in 4-hydroxyethylpiperazineethanesulfonic acid buffer solution of tris(2-carboxyethyl)phosphine for reduction to obtain reduced sericin solution;
  • Dry preparation of sericin powder place silkworm cocoons in Na 2 CO 3 solution, stir to obtain a gum silk solution, remove impurities, dialyze, and freeze-dry.
  • Tris(2-carboxyethyl)phosphine is dissolved in 4-hydroxyethylpiperazineethanesulfonic acid buffer at a ratio of 1-30g/mL
  • the preparation of the reduced sericin solution the sericin is dissolved in the buffer solution of 4-hydroxyethylpiperazineethanesulfonic acid of three (2-carboxyethyl) phosphine to make a concentration of 10-30% (W/V ) to reduce sericin solution.
  • Dry sericin powder is dissolved in tris(2-carboxyethyl)phosphine solution at a ratio of 0.01-0.3g/mL,
  • the mass ratio of sericin powder to tris(2-carboxyethyl)phosphine is 1/20-1/30
  • the reduced sericin solution and the tannic acid solution are mixed at a volume ratio of (1-1.5):(1-2).
  • the concentration of tannic acid solution is 0.05-0.5g/mL.
  • the reduced sericin solution is mixed with the tannic acid solution, and the sericin adhesive is obtained after sufficient reaction.
  • the invention provides a relatively new preparation method of sericin adhesive, which is simpler and more environment-friendly; realizes the preparation of adhesive by using sericin as a raw material; and the sericin adhesive has better adhesion performance and wound repair promotion performance.
  • Fig. 1 is the performance diagram of sericin adhesive
  • Fig. 2 is the adhesive property demonstration figure of sericin adhesive
  • Fig. 3 is the demonstration figure of the bonding performance of sericin adhesive to biological tissue
  • Figure 4 is a graph showing the healing-promoting performance of sericin adhesive in vivo.
  • Figure 5 is a diagram showing the degradation of sericin adhesive in vivo.
  • Figure 6 is a graph showing the biocompatibility of sericin adhesive.
  • the preparation method of sericin adhesive comprises the steps
  • Preparation of reduced sericin dry dissolving sericin powder in 4-hydroxyethylpiperazineethanesulfonic acid buffer solution of tris(2-carboxyethyl)phosphine for reduction to obtain reduced sericin solution;
  • Dry preparation of sericin powder place silkworm cocoons in Na 2 CO 3 solution, stir to obtain a gum silk solution, remove impurities, dialyze, and freeze-dry.
  • Tris(2-carboxyethyl)phosphine is dissolved in 4-hydroxyethylpiperazineethanesulfonic acid buffer at a ratio of 1-30g/mL
  • Dry sericin powder is dissolved in tris(2-carboxyethyl)phosphine solution at a ratio of 0.01-0.3g/mL,
  • the mass ratio of sericin powder to tris(2-carboxyethyl)phosphine is 1/20-1/30
  • the preparation of the reduced sericin solution the sericin is dissolved in the 4-hydroxyethylpiperazineethanesulfonic acid buffer solution of three (2-carboxyethyl) phosphine to make a concentration of 10-20% (W/V ) to reduce sericin solution.
  • the reduced sericin solution and the tannic acid solution are mixed at a volume ratio of (1-1.5):(1-2).
  • the concentration of tannic acid solution is 0.05-0.5g/mL.
  • the reduced sericin solution is mixed with the tannic acid solution, and the sericin adhesive is obtained after sufficient reaction.
  • step 1) Dissolve the sericin or commercial sericin obtained in step 1) in a ratio of 0.1 g/mL in 1-10 mL of tris(2-carboxyethyl)phosphine solution (4-hydroxyethylpiperazineethanesulfonic acid buffer solution, pH 7.0-7.4), reacted for 30 minutes to obtain a reduced sericin solution.
  • tris(2-carboxyethyl)phosphine solution 4-hydroxyethylpiperazineethanesulfonic acid buffer solution, pH 7.0-7.4
  • mussel foot protein In addition to the DOPA group, mussel foot protein also contains some hydrophobic amino acids, and the exposed hydrophobic groups discharge surface water molecules through hydrophobic interactions, thereby achieving continuous underwater adhesion.
  • hydrophobic amino acids cyste/cystine
  • step 1) Dissolve the sericin obtained in step 1) in a ratio of 0.1g/mL in 1-10mL of tris(2-carboxyethyl)phosphine solution (4-hydroxyethylpiperazineethanesulfonic acid buffer, pH 7.0 ⁇ 7.4), react for 30 minutes to obtain a reduced sericin solution;
  • Embodiment 2 Reduced sericin and sericin adhesive characterization detection
  • A is the solution of sericin protein before and after treatment with the reducing agent tris(2-carboxyethyl)phosphine
  • B, C, and D are the sulfhydryl content, ultraviolet absorption spectrum, and zeta potential of sericin and reduced sericin, respectively.
  • the sericin solution becomes turbid after the treatment with the reducing agent
  • the figure B shows that the sulfhydryl content in the reduced sericin molecule increases significantly, indicating that the treatment with the reducing agent tris(2-carboxyethyl)phosphine causes sericin protein
  • the breakage of the disulfide bond in the molecule leads to an increase in the content of sulfhydryl groups.
  • Figure C in Figure 1 shows that the absorption peak of the hydrophobic amino acid at 280 nm in the reduced sericin solution is enhanced, which further illustrates that the breakage of the disulfide bond causes the externalization of the hydrophobic amino acid in the protein.
  • Figure D in Figure 1 shows that the sericin solution caused by the reducing agent treatment shows that the zeta potential changes from negative to positive, indicating that the externalization of hydrophobic amino acids leads to changes in the surface potential of the protein.
  • E among Fig. 1 is the preparation process of sericin adhesive
  • F figure is the physical figure of the prepared sericin adhesive (rSer-TA), which shows that the sericin adhesive has stringing phenomenon when stretched.
  • G in Figure 1 is the Fourier transform infrared spectrum (FTIR) of sericin adhesive. Compared with sericin protein, the amide I bond of sericin adhesive shifts to 1655cm -1 at 1655cm -1 , which indicates that reducing agent treatment Can not cause the change of sericin secondary structure, in Fig.
  • FTIR Fourier transform infrared spectrum
  • H and I are the X-ray photoelectron spectrum (XPS) of sericin and sericin adhesive, and the result shows the disulphide bond (HSSH) in sericin
  • XPS X-ray photoelectron spectrum
  • the above results show that the sericin adhesive is formed based on intermolecular interactions and partial covalent interactions.
  • Figure 3C-D shows that the adhesive strength of the reduced sericin adhesive to the tissue reaches 0.1MPa, while the adhesive strength of the non-reduced sericin adhesive to the tissue is only about 0.02Mpa;
  • Figure 3E-F shows the reduced sericin after soaking in water The bonding strength of the adhesive to the tissue can still reach more than 0.05MPa, while the non-reduced sericin adhesive completely loses the adhesive to the tissue.
  • the sericin adhesive has good hemostasis and sealing effect when used in the mouse small intestine and liver hemorrhage and leakage model, which shows that the sericin adhesive is also used in the small intestine and liver hemorrhage and leakage. Has a certain potential.
  • the sericin adhesive did not cause blood cell rupture, which proved that the sericin adhesive had good blood compatibility, further indicating that the sericin adhesive had good biocompatibility.

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Abstract

本发明属于医用生物复合材料领域，具体公开了一种丝胶粘合剂的制备方法及应用，丝胶粘合剂的制备方法，包括下述步骤：还原丝胶制备：丝胶蛋白溶于三(2-羧乙基)膦溶液中还原，获得还原丝胶溶液；丝胶粘合剂制备：还原丝胶溶液与单宁酸溶液混合，获得丝胶粘合剂，丝胶粘合剂作为组织粘合剂促进创面愈合的应用，本发明提供了一种较新的丝胶粘合剂制备方法，更加简单、环保；实现以丝胶作为原料制备具有水下粘合能力的粘合剂；丝胶具有较好的粘合性能和促创面修复性能。

Description

一种丝胶粘合剂的制备方法及应用 技术领域
本发明属于医用生物复合材料领域，尤其涉及丝胶粘合剂的制备方法及应用。
背景技术
临床上伤口闭合及创伤软组织重建等涉及重新连接软组织表面的工作一般使用手术缝线进行封闭。但是手术缝合往往会导致额外的创伤、渗漏。因此，寻求容易操作、无需清除的生物可降解组织黏合剂以替代传统的手术缝合线成为当务之急，引发了研究医用粘合剂的热潮。由于水占人体的70％左右，因此大部分植入人体的生物材料都处于湿润环境，这就要求粘合剂需具备水下粘合能力。人体环境中生物材料粘附失败的原因可归结为几个因素。首先，水分子可以在生物材料与组织之间的界面上形成薄薄的水合作用层，从而阻止生物材料上的粘附基团接触组织表面。其次，水分子很容易破坏生物材料与组织之间的非共价粘合相互作用。另外，吸水后生物材料(例如水凝胶)的溶胀或收缩也会削弱它们对组织的粘附力。因此，开发能够在潮湿条件下实现有效粘附的生物材料具有重大意义。
丝胶是包裹在丝素纤维表层的一种天然大分子蛋白，长期以来由于人们对丝胶认识的不足和研究的局限性，导致每年有大量丝胶在缫丝工业中被当作废物处理，浪费了大量宝贵的天然资源，并对环境造成了严重的污染。近年来，如CN 111188194等中展示，丝胶具有良好的生物性能如低免疫原性、生物可降解性、抗氧化性、细胞黏附性， 因而丝胶对皮肤组织、血管组织、骨组织损伤具有优良的修复效果。
在海洋中，贻贝可以在不断受到海浪袭击的情况下牢固地粘附在岩石等各种基质上，为水下粘合剂的开发提供了灵感。贻贝足蛋白中的多巴基团DOPA是被认为贻贝粘附的关键，它通过非共价相互作用(包括氢键，金属配位，π-π相互作用和阳离子-π相互作用)实现粘附。为了克服水对粘合剂的破坏作用，受海洋贻贝启发，研究人员在高分子链中引入DOPA基团来制备粘合剂，但是粘合剂的制备需要复杂的预改性过程或有毒的氧化剂/金属离子，限制了这类粘合剂的潜在生物应用。因此，如何制备生物相容性较好的水下粘合剂是亟待解决的问题。
发明内容
针对上述问题，本发明提供水下丝胶粘合剂的制备方法及应用，通过调节粘合剂成份的亲疏水平衡促进组织表面排水，主要解决了现有粘合剂不具备水下粘合能力，粘合强度弱，生物相容性差等问题。
为了解决上述问题，本发明采用如下技术方案：
丝胶粘合剂的制备方法，包括下述步骤
还原丝胶制备：丝胶粉干溶于三(2-羧乙基)膦的4-羟乙基哌嗪乙磺酸缓冲液中还原，获得还原丝胶溶液；
丝胶粘合剂制备：还原丝胶溶液与单宁酸溶液混合，充分反应后获得丝胶粘合剂；
一种方式中，还包括
丝胶粉干制备：蚕茧置于Na 2CO 3溶液，搅拌得胶丝溶液，除杂， 透析，冻干。
一种方式中，还原丝胶制备中：
4-羟乙基哌嗪乙磺酸缓冲液pH为7.0-7.4，
三(2-羧乙基)膦以1-30g/mL比例溶于4-羟乙基哌嗪乙磺酸缓冲液
一种方式中，还原丝胶溶液制备：丝胶溶于三(2-羧乙基)膦的4-羟乙基哌嗪乙磺酸缓冲液中，制成浓度10-30％(W/V)还原丝胶溶液。
丝胶粉干以0.01-0.3g/mL比例溶于三(2-羧乙基)膦溶液，
丝胶粉干与三(2-羧乙基)膦质量比为1/20-1/30，
一种方式中，丝胶粘合剂制备中，还原丝胶溶液、单宁酸溶液按(1-1.5)：(1-2)体积比混合。
一种方式中，丝胶粘合剂制备中：
单宁酸溶液浓度为0.05-0.5g/mL。
一种方式中，丝胶粘合剂制备中，还原丝胶溶液与单宁酸溶液混合，充分反应后获得丝胶粘合剂。
本发明的有益效果是：
提供了一种较新的丝胶粘合剂的制备方法，更加简单、环保；实现以丝胶作为原料制备粘合剂；丝胶粘合剂具有较好粘合性能和促创面修复性能。
附图说明
图1为丝胶粘合剂的各项性能展示图；
图2为丝胶粘合剂的粘合性能展示图；
图3为丝胶粘合剂对生物组织的粘合性能展示图；
图4为丝胶粘合剂体内促愈合性能展示图。
图5为丝胶粘合剂体内降解展示图。
图6为丝胶粘合剂生物相容性展示图。
具体实施方式
下面结合附图对本发明做进一步说明：
丝胶粘合剂的制备方法，包括下述步骤
还原丝胶制备：丝胶粉干溶于三(2-羧乙基)膦的4-羟乙基哌嗪乙磺酸缓冲液中还原，获得还原丝胶溶液；
丝胶粘合剂制备：还原丝胶溶液与单宁酸溶液混合，充分反应后获得丝胶粘合剂；
一种方式中，还包括
丝胶粉干制备：蚕茧置于Na 2CO 3溶液，搅拌得胶丝溶液，除杂，透析，冻干。
一种方式中，还原丝胶制备中：
4-羟乙基哌嗪乙磺酸缓冲液pH为7.0-7.4，
三(2-羧乙基)膦以1-30g/mL比例溶于4-羟乙基哌嗪乙磺酸缓冲液
丝胶粉干以0.01-0.3g/mL比例溶于三(2-羧乙基)膦溶液，
丝胶粉干与三(2-羧乙基)膦质量比为1/20-1/30，
一种方式中，还原丝胶溶液制备：丝胶溶于三(2-羧乙基)膦的4- 羟乙基哌嗪乙磺酸缓冲液中，制成浓度10-20％(W/V)还原丝胶溶液。
一种方式中，丝胶粘合剂制备中，还原丝胶溶液、单宁酸溶液按(1-1.5)：(1-2)体积比混合。
一种方式中，丝胶粘合剂制备中：
单宁酸溶液浓度为0.05-0.5g/mL。
一种方式中，丝胶粘合剂制备中，还原丝胶溶液与单宁酸溶液混合，充分反应后获得丝胶粘合剂。
丝胶粘合剂的一些物性特征如图1中展示。
下面对其中部分制备方法做进一步说明：
1)丝胶的提取
称取蚕茧(家蚕蚕茧(白玉、皓月)，棹蚕茧(A.mylitta)或蓖麻蚕茧等)，将其剪成碎片，清洗干净，去除水分。
每克蚕茧加入20mL的0.02mol/L Na 2CO 3水溶液，在100℃条件下搅拌1小时，使丝胶溶解，得到丝胶溶液。
离心去除丝胶溶液中的杂质成分，透析12～72小时，获得澄清丝胶溶液，冻干后得到丝胶粉末。
2)还原丝胶的制备
将步骤1)中得到的丝胶或商品化的丝胶以0.1g/mL的比例溶解于1-10mL的三(2-羧乙基)膦溶液中(4-羟乙基哌嗪乙磺酸缓冲液，pH7.0～7.4)中，反应30分钟得到还原丝胶蛋白溶液。
3)单宁酸溶液的配制
称取1g单宁酸溶于10mL ddH 2O中，制成浓度为10％(W/V)的单宁酸溶液
4)丝胶粘合剂的制备
按体积比1：1将还原丝胶溶液与过单宁酸溶液混合，充分搅拌得到丝胶粘合剂，取出后用ddH 2O清洗五次得到所述丝胶粘合剂。
除了DOPA基团外，贻贝足蛋白中还含有部分疏水氨基酸，暴露出的疏水基团通过疏水作用排出表面水分子，从而实现持续水下粘合。丝胶蛋白分子骨架中存在少量富含二硫键的疏水氨基酸(半胱/胱氨酸)，基于上述理论，我们利用还原剂打开丝胶蛋白中的二硫键，暴露出疏水氨基酸，进一步与单宁酸通过氢键、范德华力和疏水相互作用形成丝胶粘合剂。通过促进表面排水，调节亲水-疏水平衡来实现有效的水下粘附。
在后续的研究中，我们在体外及体内测试了该丝胶粘合剂的粘合性能及促修复性能，发现该材料具有较好的组织粘合性能，可有效促进创面的修复，同时具有良好的生物相容性。
具体实施例如下
实施例1丝胶粘合剂的制备
步骤1)丝胶的提取
1.称取蚕茧(家蚕蚕茧(白玉、皓月)，棹蚕茧(A.mylitta)或蓖麻蚕茧等)，将其剪成碎片，用ddH 2O清洗干净，3500rpm离心5分钟去除水分；
2.每克蚕茧加入20mL的0.02mol/L Na 2CO 3水溶液，置于恒温 水浴锅在100℃条件下搅拌1小时，使丝胶溶解，得到丝胶溶液；
3.将得到的丝胶溶液转入50mL离心管中，3500rpm离心5分钟去除丝胶溶液中的杂质成分，得到澄清溶液；
4.将澄清溶液转入分子截留量为3500Da的透析袋中，于ddH 2O中透析72小时，慢速搅拌，每隔6小时换一次水；
5.将丝胶溶液转入50mL离心管中，置于液氮中速冻5分钟后放入冻干机，冻干后得到丝胶粉末，放入-20℃冰箱保存备用。
步骤2)还原丝胶的制备
将步骤1)中得到的丝胶以0.1g/mL的比例溶解于1-10mL的三(2-羧乙基)膦溶液中(4-羟乙基哌嗪乙磺酸缓冲液，pH 7.0～7.4)中，反应30分钟得到还原丝胶蛋白溶液；
步骤3)单宁酸溶液的配制
称取1g单宁酸溶于10mL ddH 2O中，制成浓度为10％(W/V)的单宁酸溶液；
步骤4)丝胶粘合剂的制备
按体积比1：1将还原丝胶溶液与过单宁酸溶液混合，充分搅拌得到丝胶粘合剂，取出后用ddH 2O清洗五次得到所述丝胶粘合剂。
实施例2还原丝胶及丝胶粘合剂表征检测
图1中A图为还原剂三(2-羧乙基)膦处理前后丝胶蛋白的溶液，B、C、D分别为丝胶和还原丝胶的巯基含量、紫外吸收光谱、zeta电位。
观察如图1中A所示，还原剂处理后丝胶蛋白溶液变浑浊，B 图显示还原丝胶分子中巯基含量显著增加，说明还原剂三(2-羧乙基)膦处理引起丝胶蛋白分子中二硫键断裂，导致巯基含量增加，图1中C图显示还原丝胶溶液在280nm处疏水氨基酸的吸收峰增强，进一步说明二硫键断裂引起蛋白中疏水氨基酸外翻。图1中D图显示还原剂处理引起丝胶蛋白溶液表明zeta电位由负变正，说明疏水氨基酸外翻导致蛋白表面电位变化。
图1中E为丝胶粘合剂的制备过程，F图为制备的丝胶粘合剂(rSer-TA)实物图，显示丝胶粘合剂在拉伸时有拉丝现象。图1中G为丝胶粘合剂的傅里叶红外光谱图(FTIR)，与丝胶蛋白相比，丝胶粘合剂在1658cm -1的酰胺I键移至1655cm -1，这表明还原剂处理不会引起丝胶蛋白二级结构的变化，图1中H和I为丝胶蛋白和丝胶粘合剂的X射线光电子能谱图(XPS)，结果显示丝胶中二硫键(HSSH)的峰位于164.2eV，而丝胶粘合剂rSer-TA中S—C＝O结构峰位于168.3eV，这表明单宁酸中部分氧化的酚醛结构与丝胶蛋白中的巯基发生反应，形成迈克尔型加合物，以上结果说明该丝胶粘合剂是基于分子间相互作用和部分共价作用形成的。
实施例3丝胶粘合剂的水下粘合能力测试
一、实验过程
1.准备1cm*3cm陶瓷片，将还原丝胶粘合剂和非还原丝胶粘合剂分别涂抹在陶瓷片上，随后放入水中；
2.将100g砝码放置到陶瓷片上，等待30s，待其粘合；
3.提起砝码，观察界面粘合情况及陶瓷片脱落情况。
二、结果分析
如图2A所示，提起砝码时，还原丝胶粘合剂将砝码和陶瓷片粘合在一起，而且流水冲击30s后二者仍然粘合在一起(图2B)，说明还原丝胶粘合剂具有水下粘合能力。而非还原丝胶粘合剂无法将砝码和陶瓷片粘合，说明其不具备水下粘合能力。以上结果说明还原丝胶粘合剂由于具有一定的疏水基团有助于增强其水下粘合性能。
实施例4丝胶粘合剂体外粘合能力测试
一、实验过程
用还原丝胶粘合剂粘合两块基底片(聚甲基丙烯酸甲酯PMMA片，铁片，硅片，陶瓷片)，待固化30min后用力学拉伸仪检测粘合剂的粘合强度。
二、结果分析
1.如图2中C-D所示，丝胶粘合剂粘合的基底片粘合强度均达到0.5MPa以上，表现出很强的粘合强度。
2.如图2中E-F所示，随着单宁酸含量增加，丝胶粘合剂的粘合强度呈递增形式。
实施例5丝胶粘合剂对组织粘合能力测试
一、实验过程
1.准备猪皮，小肠和大肠组织裁剪成一定尺寸；
2.将还原丝胶粘合剂和非还原丝胶粘合剂分别涂抹在三种组织表面，通过扭转组织观察粘合剂的粘合强度，并进一步将组织贴附在基底片上检测力学粘合强度；
3.将粘合的组织浸泡在水中30min，取出后通过扭转观察粘合剂的粘合强度，并进一步检测力学粘合强度；
二、结果分析
如图3A所示，还原丝胶粘合剂粘合的组织在大力扭转时仍然粘附在组织表面，浸水30min后粘合剂仍保留在组织表面，且在大力扭转时不会脱落。图3B显示非还原粘合剂粘合的组织在大力扭转时显示出有缝隙存在，浸水30min后粘合剂脱落。以上结果说明疏水基团外翻导致还原丝胶粘合剂对组织的粘合强度更高，进一步说明调节粘合剂的亲疏水平衡可调节其水下粘合能力。
如图3C-D显示还原丝胶粘合剂对组织的粘合强度达到0.1MPa，而非还原丝胶粘合剂对组织的粘合强度仅为约0.02Mpa；图3E-F显示浸水后还原丝胶粘合剂对组织的粘合强度仍能达到0.05MPa以上，而非还原丝胶粘合剂对组织的粘合剂完全丧失。
实施例6丝胶粘合剂对组织渗漏的封堵能力测试
一、实验过程
1.准备一段大肠，装入水并将两端系上，用注射器戳一个小孔，水可从小孔身处；
2.取粘合剂封堵小孔，观察渗漏情况。
二、结果分析
如图3G所示，大肠组织由于穿孔出现渗漏，将还原丝胶粘合剂涂抹在渗漏口部位，按压5s，粘合剂贴在组织表面，同时渗漏被封堵；
以上结果说明还原丝胶粘合剂对组织渗漏具有一定的封堵能力。
实施例7丝胶粘合剂动物创面粘合测试
一、丝胶粘合剂小鼠肝部止血测试
如图4中A所示，剪开小鼠腹部皮肤，暴露出肝脏，用生理盐水擦拭干净，将垫有滤纸的培养皿置于肝脏下面，将注射器扎入肝组织，血液流出，将粘合剂涂抹在出血部位，观察伤口出血情况，3M粘合剂组作为对照。
二、丝胶粘合剂大鼠创面愈合模型愈合测试
如图4中D所示，用剪刀在250g左右SD大鼠背部剪开2cm伤口，用医用无菌纱布将周围渗血擦掉，将还原丝胶粘合剂涂抹在表皮侧面，粘合30s，观察创面粘合情况。并继续饲养14天，记录创面愈合情况。以手术缝合的动物和3M粘合剂粘合的动物作为对照。
三、丝胶粘合剂对大鼠体内小肠渗漏封堵止血测试
如图4中K所示，剪开大鼠腹部皮肤，找到小肠，用医用无菌纱布将周围液体吸尽后，将注射器扎入小肠组织，体液流出，将粘合剂涂抹在出血部位，观察创口体液渗漏情况，不处理组作为阴性对照。
四、结果分析
1.如图4中B、C所示，丝胶粘合剂用于鼠肝损伤出血模型出血时间和出血量明显低于对照，证明丝胶粘合剂具有良好的止血效果，说明材料在内脏器官出血止血方面具有一定作用。
2.如图4中E、F所示，丝胶粘合剂用于创面愈合实验，丝胶粘合剂组伤口愈合率与对照组显著高于对照组，图G-J显示，丝胶粘合 剂组创面愈合质量较好，有毛囊和皮脂腺和汗腺生成，明显优于对照组。显示出丝胶粘合剂用于创面愈合的优越性。
3.如图4中K、L所示，丝胶粘合剂用于鼠小肠、肝出血渗漏模型具有良好的止血、封堵效果，说明丝胶粘合剂用于小肠、肝出血渗漏方面同样具有一定潜力。
实施例8丝胶粘合剂体内降解
一、实验过程
1.剪开大鼠背部皮肤，将粘合剂涂抹在背部组织，缝合组织观察7天。
2.分别于第3天、第5天、第7天打开伤口，观察粘合剂降解情况。
二、结果分析
如图5A所示，丝胶粘合剂在第三天时被组织包裹，出现一定的炎症反应，第5天粘合剂变小，第7天完全降解，说明丝胶粘合剂在体内具有可降解性。图5B切片染色结果说明粘合剂没有明显的炎症反应和毒性。
一、结果分析
如图6B所示，丝胶粘合剂不会导致血细胞破裂，证明丝胶粘合剂具有良好的血液相容性，进一步说明丝胶粘合剂具有良好的生物相容性。
最后应说明的是：以上仅为本发明的优选实例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本 领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (6)

  1. 一种丝胶粘合剂的制备方法，包括还原丝胶制备和丝胶粘合剂制备，其特征在于：还原丝胶制备和丝胶粘合剂制备的步骤如下：
    还原丝胶制备：丝胶粉干溶于三(2-羧乙基)膦的4-羟乙基哌嗪乙磺酸缓冲液中还原，获得还原丝胶溶液；
    丝胶粘合剂制备：还原丝胶溶液与单宁酸溶液混合，充分反应后获得丝胶粘合剂。
  2. 根据权利要求1所述的一种丝胶粘合剂的制备方法，其特征在于：丝胶粉干制备：蚕茧置于Na 2CO 3溶液，搅拌得胶丝溶液，除杂，透析，冻干。
  3. 根据权利要求1所述的一种丝胶粘合剂的制备方法，其特征在于：还原丝胶的制备方法如下：
    4-羟乙基哌嗪乙磺酸缓冲液pH为7.0-7.4；
    三(2-羧乙基)膦以1-30g/mL比例溶于4-羟乙基哌嗪乙磺酸缓冲液；
    丝胶粉干以0.01-0.3g/mL比例溶于三(2-羧乙基)膦溶液。
  4. 根据权利要求1所述的一种丝胶粘合剂的制备方法，其特征在于：丝胶粘合剂的制备方法如下，其中单宁酸溶液制备：单宁酸溶于水中，制成浓度5-50％单宁酸溶液。
  5. 根据权利要求1所述的一种丝胶粘合剂的制备方法，其特征在于：丝胶粘合剂的制备方法中还原丝胶溶液、单宁酸溶液按(1-1.5)：(1-2)体积比混合。
  6. 一种丝胶粘合剂的应用，其特征在于：丝胶粘合剂在制备止血、 创面粘合材料中的应用。
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CN112843325A (zh) * 2021-01-20 2021-05-28 江苏德威兰医疗器械股份有限公司 一种医用水凝胶粘合剂及其制备方法和用途
CN113398323A (zh) * 2021-06-30 2021-09-17 华中科技大学同济医学院附属协和医院 一种丝胶粘合剂的制备方法及应用

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