WO2021208688A1 - 一种抗肠粘连双层生物凝胶制备方法及双层生物凝胶 - Google Patents

一种抗肠粘连双层生物凝胶制备方法及双层生物凝胶 Download PDF

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WO2021208688A1
WO2021208688A1 PCT/CN2021/082637 CN2021082637W WO2021208688A1 WO 2021208688 A1 WO2021208688 A1 WO 2021208688A1 CN 2021082637 W CN2021082637 W CN 2021082637W WO 2021208688 A1 WO2021208688 A1 WO 2021208688A1
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solution
layer
biogel
aema
chitosan
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French (fr)
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黄文华
吴耀彬
伍笑棋
许益超
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南方医科大学
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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
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • A61L31/06—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • 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
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • A61L31/042—Polysaccharides
    • 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
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified 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
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/145—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
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/04—Materials for stopping bleeding

Definitions

  • the invention relates to the field of biomedicine, in particular to a method for preparing a double-layer biogel for anti-intestinal adhesion and a double-layer biogel.
  • Intestinal adhesion is a common surgical system disease, which is abnormal adhesion between the intestine and the intestine or between the intestine and the internal organs of the abdominal cavity.
  • partial adhesions may restrict the peristalsis of the intestines or pull the intestines, leading to long-term paroxysmal abdominal pain or bloating; when the intestinal adhesions cause incomplete intestinal obstruction, although the food can pass through the intestines at this time, the adhesions It will cause food to pass through the intestines here slowly, and patients will experience nausea and paroxysmal abdominal pain and bloating after meals; when intestinal adhesions have complete intestinal obstruction, patients will experience abdominal pain, nausea, and stop exhausting. symptom.
  • intestinal adhesions The main cause of intestinal adhesions is abdominal surgery.
  • conservative treatment that is, controlling diet, taking drugs and strengthening exercise, which can relieve the pain caused by intestinal adhesions but cannot relieve intestinal adhesions; the other is to be able to relieve intestinal adhesions.
  • Second surgery for intestinal adhesions but the second operation may produce new intestinal adhesions.
  • the problem of intestinal adhesions has not yet been resolved in the medical field.
  • One of the objectives of the present invention is to avoid the deficiencies of the prior art and provide a method for preparing an anti-intestinal adhesion double-layer biogel, which can prepare an anti-intestinal adhesion double-layer biogel, which can act on Between the abdominal cavity and the intestine, and between the intestine and the intestine, a physical barrier is formed to block the occurrence of intestinal adhesions.
  • a preparation method of anti-intestinal adhesion double-layer biological gel includes the following steps:
  • the methacrylic anhydride solution is added dropwise to the chitosan solution, and the mixture formed by the reaction is dialyzed and lyophilized to obtain pure methacrylated chitosan;
  • the pure product of methacrylated chitosan is prepared into a solution with a mass volume concentration of 2-10% w/v in 5-50 ml, a photoinitiator is added, and the mixture is stirred uniformly, and then stored at 4°C;
  • the pure HA-DA-AEMA product is prepared into 5-50ml of a solution with a mass volume concentration of 2-10% w/v, the photoinitiator is added, and the mixture is stirred uniformly, and then stored at 4°C;
  • HA-DA-AEMA solution place the HA-DA-AEMA solution on a flat dish, add a photoinitiator, and irradiate it with a 365nm ultraviolet light for 30 to 90 seconds;
  • HA-DA-AEMA solution changes from liquid to liquid-solid
  • methacrylated chitosan solution on the HA-DA-AEMA liquid-solid
  • add photoinitiator and irradiate it with 365nm UV light for 30 ⁇ 150s, until the two solutions all become solid, forming an anti-intestinal adhesion double-layer biogel composed of an inner layer biogel that adheres to the tissue and an anti-adhesion outer layer biogel.
  • the mass volume concentration of the methacrylated chitosan solution in S3 is 4 to 7% w/v
  • the mass volume ratio of the S4 sodium hyaluronate solution is 7% w/v to 15% w/v
  • S5 The mass volume concentration of the HA-DA-AEMA solution in the medium is 4 to 7% w/v.
  • the dropwise addition of methacrylic anhydride to S2 is specifically: stirring and heating the chitosan solution in an oil bath to 50 °C ⁇ 60 °C, and the concentration of 94%-95% methacrylic anhydride at 0.5-1ml/min The speed was added dropwise to the chitosan solution and stirred with a magnetic stirrer at a speed of 1000r/min and stirred for 4-6h.
  • the dialysis and freeze-drying in S2 is specifically: placing the mixture in a dialysis bag for distilled water dialysis for 5-6 days at room temperature. After the dialysis is completed, the mixture is placed at a temperature of -40°C to 80°C and a pressure of 5-100 mbar. Freeze drying for 24 to 48 hours.
  • dialysis and freeze-drying in S2 is specifically: placing the container carrying the mixed solution on a magnetic stirrer at room temperature, adjusting the speed of the stirrer to 850 ⁇ 1000r/min, and stirring for 24 ⁇ 48h;
  • the mixed solution is placed in a dialysis bag and dialyzed with PBS for 3 to 5 days. After the dialysis is completed, the mixture is freeze-dried at a temperature of -80° C. and a pressure of 300 to 400 mbar for 24 to 48 hours.
  • the photoinitiator is Irgacure 2959 or LAP, and the mass volume fraction of Irgacure 2959 and LAP are both 0.1-20% w/v.
  • the deacetylation degree of the chitosan in S1 ranges from 75% to 90%, the molecular mass is from 310000 to 375000 g ⁇ mol-1, and the viscosity is from 800 to 2000.
  • the molecular weight of sodium hyaluronate in S4 is 90,000 to 100,000
  • the molecular weight of dopamine is 189.64
  • the molecular weight of methacrylic acid-2-aminoethyl ester is 165.62.
  • the inner layer of biogel that adheres to the tissue can be tightly combined with the surface of the intestine, can play a role in fixing the intestinal wound, prevent inflammatory exudation and bleeding, and the anti-adhesion outer layer of biogel can avoid adhesion to the tissue
  • the inner biogel adhesive surface adheres to the abdominal cavity or other intestines, avoiding the pain caused by intestinal adhesions to the patient.
  • Another purpose of the present invention is to avoid the shortcomings of the prior art and provide a double-layer biogel, which can act on the abdominal cavity and the intestine, between the intestine and the intestine, to form a physical barrier and block the occurrence of intestinal adhesions. .
  • a double-layer biological gel is prepared by adopting a preparation method of anti-intestinal adhesion double-layer biological gel.
  • the double-layer biological gel provided by the present invention is prepared by the preparation method of the anti-intestinal adhesion double-layer biological gel.
  • the preparation method of the anti-intestinal adhesion double-layer biogel includes the following steps: S1: preparing chitosan solution in advance; S2: preparing pure methacrylated chitosan; S3: preparing methacrylated chitosan solution ; S4: preparation of pure HA-DA-AEMA; S5: preparation of HA-DA-AEMA solution; S6: curing of HA-DA-AEMA solution and methacrylated chitosan solution to adhere to the tissue Layer biogel and anti-adhesion outer biogel.
  • the inner layer of biogel that adheres to the tissue can be tightly combined with the surface of the intestine, can play a role in fixing the intestinal wound, prevent inflammatory exudation and bleeding, and the anti-adhesion outer layer of biogel can avoid adhesion to the tissue
  • the inner biogel adhesive surface adheres to the abdominal cavity or other intestines, avoiding the pain caused by intestinal adhesions to the patient.
  • Figure 1 is a schematic diagram of the synthesis mechanism of the anti-adhesion outer layer gel of the present invention.
  • Figure 2 is a schematic diagram of the synthesis mechanism of the inner layer gel of the present invention and tissue adhesion.
  • Figure 3 is a schematic diagram of the combination of the outer layer biogel and the inner layer biogel of the present invention into a bilayer structured biogel.
  • a preparation method of anti-intestinal adhesion double-layer biological gel includes the following steps:
  • the specific preparation of chitosan solution in S1 is: dissolving chitosan with a mass volume fraction of 3% w/v in 1% acetic acid, stirring with a magnetic stirrer, and adjusting the speed of the stirrer to 1000r/min. Stir for 24h. It should be noted that the mixed solution of chitosan and acetic acid should be shaken evenly at room temperature before being stirred with a magnetic stirrer. In addition, the volume of chitosan can be calculated based on the mass-to-volume ratio of 3% w/v, which is the basis Those in the field are familiar with the process, so I won't repeat it here.
  • the methacrylic anhydride solution is added dropwise to the chitosan solution, and the mixture formed by the reaction is dialyzed and lyophilized to obtain pure methacrylated chitosan;
  • the specific steps of adding methacrylic anhydride in S2 are: stirring and heating the chitosan solution in an oil bath to 50°C, adding 94% methacrylic anhydride to the chitosan solution at a speed of 0.5, and stirring with a magnetic force. Stirring is carried out with a rotating speed of 1000r/min and stirring for 4h.
  • the mass volume of methacrylic anhydride can be calculated from the mass volume of chitosan in S1 and the mass volume concentration of the methacrylated chitosan solution in S3. This is a process well known to those in the art. This will not be repeated here.
  • the specific dialysis freeze-drying in S2 is: placing the mixture in a dialysis bag for distilled water dialysis for 5 days at room temperature. After the dialysis is completed, the mixture is freeze-dried at a temperature of -40° C. and a pressure of 5 mbar for 24 hours.
  • the dialysis bag used in the dialysis in this example is a dialysis bag with a molecular weight of 12-14 kDa from Sigma in the United States.
  • methacrylic anhydride solution is added dropwise to the chitosan solution, as shown in Figure 1, the chitosan will react with methacrylic anhydride to form methacrylated chitosan. After the chitosan is completely reacted, the mixture should be methacrylated chitosan and residual methacrylic anhydride, so use a dialysis bag to filter out the residual methacrylic anhydride to obtain pure methacrylated chitosan .
  • the pure product of methacrylated chitosan is prepared into 5-50ml solution with a mass volume concentration of 2% w/v, and the photoinitiator is added and stirred uniformly, and then stored at 4°C; this embodiment After stirring for 2 hours, the methacrylated chitosan solution and the initiator were mixed uniformly. It should be noted that the amount of pure methacrylated chitosan can be calculated based on the mass volume concentration of the methacrylated chitosan solution. This process is well known to those in the art and will not be repeated here. .
  • the specific dialysis freeze-drying in S4 is: at room temperature, place the container carrying the mixed solution on a magnetic stirrer, adjust the speed of the stirrer to 850r/min, and stir for 24h;
  • the mixed solution was placed in a dialysis bag and dialyzed with PBS for 3 days. After the dialysis, the mixture was freeze-dried at a temperature of -80° C. and a pressure of 300 mbar for 24 hours.
  • the molecular mass of sodium hyaluronate is 90,000
  • the molecular mass of dopamine is 189.64
  • the molecular mass of methacrylic acid-2-aminoethyl ester acid is 165.62.
  • the pure HA-DA-AEMA product is prepared into 5-50ml solution with a mass volume concentration of 2% w/v, and the photoinitiator is added and stirred evenly, and then stored at 4°C;
  • HA-DA-AEMA solution place the HA-DA-AEMA solution on a flat dish, add a photoinitiator, and irradiate it with a 365nm ultraviolet light for 30 to 90 seconds;
  • the method for preparing the anti-intestinal adhesion double-layer biogel uses ultraviolet light cross-linking to make the anti-adhesion outer layer biogel composed of methacrylated chitosan and HA-DA-AEMA to adhere to tissues
  • the inner layer of biogel is chemically cross-linked and tightly combined into a whole.
  • the formed double-layer biogel can not only fix the intestinal wound, prevent inflammatory exudation and bleeding, but also avoid the sticky surface and The walls of the abdominal cavity or the intestines are joined to prevent unnecessary adhesions.
  • a preparation method of anti-intestinal adhesion double-layer biogel The other features are the same as that of Example 1, except that: the pre-prepared chitosan solution in S1 is specifically: a shell with a mass volume fraction of 10% w/v The glycan was dissolved in 5% acetic acid and stirred with a magnetic stirrer at a speed of 1000r/min for 48h.
  • the chitosan described in S1 has a deacetylation degree range of 90%, a molecular mass of 375,000 g ⁇ mol-1, and a viscosity of 2,000.
  • the specific steps of adding methacrylic anhydride in S2 are: stirring and heating the chitosan solution in an oil bath to 60°C, and adding 95% methacrylic anhydride to the chitosan solution at a rate of 1ml/min. Stir with a magnetic stirrer at a speed of 1000r/min for 6h.
  • the mass volume concentration of the methacrylated chitosan solution in S3 is 10% w/v.
  • the specific dialysis freeze-drying in S4 is: at room temperature, place the container carrying the mixed solution on a magnetic stirrer, adjust the speed of the stirrer to 1000r/min, and stir for 48h;
  • the molecular mass of sodium hyaluronate is 100,000
  • the molecular mass of dopamine is 189.64
  • the molecular mass of methacrylic acid-2-aminoethyl ester is 165.62.
  • the mass volume concentration of HA-DA-AEMA solution in S5 is 10% w/v.
  • Both Irgacure 2959 and LAP have a mass volume fraction of 20% w/v.
  • the method for preparing the anti-intestinal adhesion double-layer biogel uses ultraviolet light cross-linking to make the anti-adhesion outer layer biogel composed of methacrylated chitosan and HA-DA-AEMA to adhere to tissues
  • the inner layer of biogel is chemically cross-linked and tightly combined into a whole.
  • the formed double-layer biogel can not only fix the intestinal wound, prevent inflammatory exudation and bleeding, but also avoid the sticky surface and The walls of the abdominal cavity or the intestines are joined to prevent unnecessary adhesions.
  • a preparation method of anti-intestinal adhesion double-layer biogel The other features are the same as in Example 1, except that: the pre-prepared chitosan solution in S1 is specifically: a shell with a mass volume fraction of 5% w/v The glycan was dissolved in 3% acetic acid and stirred with a magnetic stirrer. The speed of the stirrer was adjusted to 1000r/min and stirred for 36h.
  • the chitosan described in S1 has a deacetylation degree range of 83%, a molecular mass of 350,000 g ⁇ mol-1, and a viscosity of 1,500.
  • the specific steps of adding methacrylic anhydride in S2 are: stirring and heating the chitosan solution in an oil bath to 55°C, adding 94.5% methacrylic anhydride to the chitosan solution at a rate of 0.75ml/min. Stir with a magnetic stirrer at a speed of 1000r/min and stir for 5h.
  • the mass volume concentration of the methacrylated chitosan solution in S3 is 6% w/v.
  • the specific dialysis freeze-drying in S4 is: at room temperature, place the container carrying the mixed solution on a magnetic stirrer, adjust the speed of the stirrer to 920r/min, and stir for 36h;
  • the mixed solution was placed in a dialysis bag and dialyzed with PBS for 4 days. After the dialysis, the mixture was freeze-dried at a temperature of -80° C. and a pressure of 350 mbar for 36 hours.
  • the molecular mass of sodium hyaluronate is 95,000
  • the molecular mass of dopamine is 189.64
  • the molecular mass of methacrylic acid-2-aminoethyl ester is 165.62.
  • the mass volume concentration of HA-DA-AEMA solution in S5 is 6% w/v.
  • Both Irgacure 2959 and LAP have a mass volume fraction of 0.1-20% w/v.
  • the method for preparing the anti-intestinal adhesion double-layer biogel uses ultraviolet light cross-linking to make the anti-adhesion outer layer biogel composed of methacrylated chitosan and HA-DA-AEMA to adhere to tissues
  • the inner layer of biogel is chemically cross-linked and tightly combined into a whole.
  • the formed double-layer biogel can not only fix the intestinal wound, prevent inflammatory exudation and bleeding, but also avoid the sticky surface and The walls of the abdominal cavity or the intestines are joined to prevent unnecessary adhesions.
  • a preparation method of anti-intestinal adhesion double-layer biogel is the same as in Example 1, except that the mass volume concentration of the methacrylated chitosan solution in S3 is 4 to 7% w/v, The mass volume ratio of the S4 sodium hyaluronate solution is 7% w/v to 15% w/v, and the mass volume concentration of the HA-DA-AEMA solution in S5 is 4 to 7% w/v.
  • the method for preparing the anti-intestinal adhesion double-layer biogel uses ultraviolet light cross-linking to make the anti-adhesion outer layer biogel composed of methacrylated chitosan and HA-DA-AEMA to adhere to tissues
  • the inner layer of biogel is chemically cross-linked and tightly combined into a whole.
  • the formed double-layer biogel can not only fix the intestinal wound, prevent inflammatory exudation and bleeding, but also avoid the sticky surface and The walls of the abdominal cavity or the intestines are joined to prevent unnecessary adhesions.
  • a double-layer biological gel is prepared by adopting a preparation method of anti-intestinal adhesion double-layer biological gel.
  • the preparation method of the anti-intestinal adhesion double-layer biogel includes the following steps: S1: preparing chitosan solution in advance; S2: preparing pure methacrylated chitosan; S3: preparing methacrylated chitosan solution ; S4: preparation of pure HA-DA-AEMA; S5: preparation of HA-DA-AEMA solution; S6: curing of HA-DA-AEMA solution and methacrylated chitosan solution to adhere to the tissue Layer biogel and anti-adhesion outer biogel.
  • double-layer biogel after abdominal surgery Take the use of double-layer biogel after abdominal surgery as an example to illustrate the use of double-layer biogel: apply HA-DA-AEMA solution to the surgical incision and surgically expose the surface of the intestine after the abdominal surgery is completed but before the wound is sutured. , Gastrointestinal anastomosis, etc., irradiate with 365nm ultraviolet light for 30s to convert it from liquid to liquid-solid to form an inner layer of biogel that adheres to the tissue, and then coat the methacrylated chitosan solution on the tissue.
  • the attached inner layer of biogel is irradiated with 365nm ultraviolet light for 60s, after both the HA-DA-AEMA solution and the methacrylate chitosan solution are solidified, the double layer gel is formed, and the wound is sutured. Among them, the methacrylated chitosan solution solidifies into an anti-adhesion outer layer biogel.
  • the double-layer biogel the inner biogel that adheres to the tissue can be tightly combined with the surface of the intestine, can play a role in fixing the intestinal wound, prevent inflammatory exudation and bleeding, and resist adhesion of the outer biogel It can avoid the adhesion of the inner biogel adhesive surface that adheres to the tissue and the abdominal cavity or other intestines, and avoids the pain caused by the intestinal adhesion to the patient.

Abstract

本发明提供的一种抗肠粘连双层生物凝胶的制备方法，包括以下步骤：S1：预先制备壳聚糖溶液；S2：制备甲基丙烯酸酯化壳聚糖纯品；S3：制备甲基丙烯酸酯化壳聚糖溶液；S4：制备HA-DA-AEMA纯品；S5：HA-DA-AEMA溶液制备；S6：将HA-DA-AEMA溶液和甲基丙烯酸酯化壳聚糖溶液分别固化成与组织粘附的内层生物凝胶和抗粘连的外层生物凝胶。与组织粘附的内层生物凝胶可以和肠道表面紧密结合，能够起到固定肠道伤口的作用，防止炎性渗出和出血，抗粘连外层生物凝胶可以避免与组织粘附的内层生物凝胶粘性面和腹腔或者和其它肠道粘连，避免了肠粘连给患者带来的痛苦。

Description

一种抗肠粘连双层生物凝胶制备方法及双层生物凝胶 技术领域
本发明涉及生物医学领域，特别是涉及一种抗肠粘连双层生物凝胶的制备方法及双层生物凝胶。
背景技术
肠粘连是一种常见的外科系统疾病，是肠管与肠管之间或者肠管与腹腔内脏器之间发生不正常的粘连。
肠粘连形成后，部分粘连可能会限制肠管的蠕动或者牵拉肠管，导致患者长期阵发性的轻微腹痛或者腹胀；当肠粘连引起不全性肠梗阻时，尽管此时食物可以通过肠管，但是粘连会导致食物通过此处肠管的速度缓慢，患者餐后会出现恶心和阵发性的腹痛和腹胀；当肠粘连出现完全性肠梗阻时，患者会出现腹痛、恶心和排气排气便停止等症状。
导致肠粘连发生的主要原因为腹部手术。现有技术中，肠粘连的解决办法有两种，一种为保守治疗，即控制饮食、服用药物和加强运动，能缓解肠粘连带来的疼痛但不能解除肠粘连；另一种为能够解除肠粘连的二次手术，但二次手术可能会产生新的肠粘连。肠粘连问题目前在医学界尚未得到解决。
因此，针对现有技术不足，提供一种抗肠粘连双层生物凝胶的制备方法及双层生物凝胶以克服现有技术不足甚为必要。
发明内容
本发明的目的之一在于避免现有技术的不足之处而提供一种抗肠粘连双层生物凝胶的制备方法，能够制备出抗肠粘连的双层生物凝胶，该凝胶能够作用于腹腔与肠道、肠道与肠道之间，形成物理屏障，阻断肠粘连发生。
本发明的上述目的通过以下技术措施实现：
提供一种抗肠粘连双层生物凝胶的制备方法，包括以下步骤：
S1：预先制备壳聚糖溶液；
S2：制备甲基丙烯酸酯化壳聚糖纯品；
具体是，在壳聚糖溶液中滴加甲基丙烯酸酐溶液，将反应形成的混合物透析冻干获得甲基丙烯酸酯化壳聚糖纯品；
S3：制备甲基丙烯酸酯化壳聚糖溶液；
具体是，取甲基丙烯酸酯化壳聚糖纯品配制成5～50ml质量体积浓度为2～10％w/v的溶液，加入光引发剂后搅拌均匀，然后置于4℃下保存；
S4：制备HA-DA-AEMA纯品；
具体是，分别称量4～5mmol的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、4～5mmol的N-羟基琥珀酰亚胺、2～5mmol的多巴胺和2～3mmol的甲基丙烯酸-2-氨基乙基酯酸置入避光离心管中；
将5～50ml质量体积比为1％w/v～20％w/v透明质酸钠溶液置于容器中，使用0.1～0.3mol/L盐酸调节透明质酸钠溶液溶液PH值至5.0～6.0，然后加入称量好的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐并搅拌20～30min，然后依次加入称量好的N-羟基琥珀酰亚胺、多巴胺和甲基丙烯酸-2-氨基乙基酯酸获得混合溶液，透析冻干后获得HA-DA-AEMA纯品；
S5：制备HA-DA-AEMA溶液；
具体是，取HA-DA-AEMA纯品配制成5～50ml质量体积浓度为2～10％w/v的溶液，加入光引发剂后搅拌均匀，然后置于4℃下保存；
S6：将HA-DA-AEMA溶液和甲基丙烯酸酯化壳聚糖溶液分别固化成与组织粘附的内层生物凝胶和抗粘连的外层生物凝胶；
具体是，将HA-DA-AEMA溶液平铺皿上，加入光引发剂，用365nm紫外灯光照射30～90s；
待HA-DA-AEMA溶液从液态变为液-固态后将甲基丙烯酸酯化壳聚糖溶液平铺到HA-DA-AEMA液-固态上，加入光引发剂，然后用365nm紫外灯光照射 30～150s，直至两种溶液全部变为固态，由与组织粘附的内层生物凝胶和抗粘连的外层生物凝胶构成的抗肠粘连双层生物凝胶形成。
优选的，S1中预先制备壳聚糖溶液具体是：将质量体积分数为3％w/v～10％w/v的壳聚糖溶于50～200ml浓度为1％～5％醋酸中，用磁力搅拌器进行搅拌，调节搅拌器转速为1000r/min，搅拌24～48h。
优选的，S3中甲基丙烯酸酯化壳聚糖溶液质量体积浓度为4～7％w/v，S4透明质酸钠溶液的质量体积比为7％w/v～15％w/v，S5中HA-DA-AEMA溶液质量体积浓度为4～7％w/v。
优选的，S2中滴加甲基丙烯酸酐具体是：在油浴锅中搅拌加热壳聚糖溶液至50℃～60℃，将浓度为94％～95％甲基丙烯酸酐以0.5～1ml/min速度滴加于壳聚糖溶液中，用磁力搅拌器进行搅拌，转速为1000r/min，搅拌4～6h。
优选的，S2中透析冻干具体是：在室温下，将混合物置于透析袋中进行蒸馏水渗析5～6d，渗析结束后，将混合物在-40℃～80℃温度下、5～100mbar压力下冷冻干燥24～48h。
优选的，S2中透析冻干具体是：在常温下，将承载混合溶液的容器置于磁力搅拌器上，调节搅拌器转速为850～1000r/min，搅拌24～48h；
搅拌结束后，将混合溶液置于透析袋中，用PBS透析3～5天，透析结束后，将混合物在-80℃温度下、300～400mbar压力下冷冻干燥24～48h。
优选的，光引发剂为Irgacure 2959或者LAP，Irgacure 2959和LAP质量体积分数都为0.1～20％w/v。
优选的，S1中所述壳聚糖的脱乙酰度范围为75～90％，分子质量为310000～375000g·mol-1，粘度为800-2000。
优选的，S4中所述透明质酸钠分子质量为9万～10万，多巴胺分子质量为189.64，甲基丙烯酸-2-氨基乙基酯酸分子质量为165.62。
本发明提供的一种抗肠粘连双层生物凝胶的制备方法，包括以下步骤：S1：预先制备壳聚糖溶液；S2：制备甲基丙烯酸酯化壳聚糖纯品；S3：制备甲基丙 烯酸酯化壳聚糖溶液；S4：制备HA-DA-AEMA纯品；S5：HA-DA-AEMA溶液制备；S6：将HA-DA-AEMA溶液和甲基丙烯酸酯化壳聚糖溶液分别固化成与组织粘附的内层生物凝胶和抗粘连的外层生物凝胶。与组织粘附的内层生物凝胶可以和肠道表面紧密结合，能够起到固定肠道伤口的作用，防止炎性渗出和出血，抗粘连外层生物凝胶可以避免与组织粘附的内层生物凝胶粘性面和腹腔或者和其它肠道粘连，避免了肠粘连给患者带来的痛苦。
本发明的另一目的在于避免现有技术的不足之处而提供一种双层生物凝胶，能够作用于腹腔与肠道、肠道与肠道之间，形成物理屏障，阻断肠粘连发生。
本发明的上述目的通过以下技术措施实现：
一种双层生物凝胶，采用抗肠粘连双层生物凝胶的制备方法制备。
本发明提供的一种双层生物凝胶，采用抗肠粘连双层生物凝胶的制备方法制备。抗肠粘连双层生物凝胶的制备方法包括以下步骤：S1：预先制备壳聚糖溶液；S2：制备甲基丙烯酸酯化壳聚糖纯品；S3：制备甲基丙烯酸酯化壳聚糖溶液；S4：制备HA-DA-AEMA纯品；S5：HA-DA-AEMA溶液制备；S6：将HA-DA-AEMA溶液和甲基丙烯酸酯化壳聚糖溶液分别固化成与组织粘附的内层生物凝胶和抗粘连的外层生物凝胶。与组织粘附的内层生物凝胶可以和肠道表面紧密结合，能够起到固定肠道伤口的作用，防止炎性渗出和出血，抗粘连外层生物凝胶可以避免与组织粘附的内层生物凝胶粘性面和腹腔或者和其它肠道粘连，避免了肠粘连给患者带来的痛苦。
说明书附图
利用附图对本发明作进一步的说明，但附图中的内容不构成对本发明的任何限制。
图1是本发明抗粘连外层凝胶合成机理示意图。
图2是本发明与组织粘附内层凝胶合成机理示意图。
图3是本发明外层生物凝胶和内层生物凝胶结合为双层结构生物凝胶的示 意图。
具体实施方式
结合以下实施例对本发明作进一步说明。
实施例1。
一种抗肠粘连双层生物凝胶的制备方法，包括以下步骤：
S1：预先制备壳聚糖溶液；
S1中预先制备壳聚糖溶液具体是：将质量体积分数为3％w/v的壳聚糖溶于浓度为1％醋酸中，用磁力搅拌器进行搅拌，调节搅拌器转速为1000r/min，搅拌24h。需要说明的是，壳聚糖与醋酸混合溶液用磁力搅拌器进行搅拌之前应再室温下震荡均匀，另外，壳聚糖的体积可以根据质量体积比3％w/v来推算出，此为本领域人员熟知过程，在此不再赘述。
S1中所述壳聚糖的脱乙酰度范围为75％，分子质量为310000g·mol-1，粘度为800。壳聚糖脱乙酰度越高越有利于壳聚糖溶解。
S2：制备甲基丙烯酸酯化壳聚糖纯品；
具体是，在壳聚糖溶液中滴加甲基丙烯酸酐溶液，将反应形成的混合物透析冻干获得甲基丙烯酸酯化壳聚糖纯品；
S2中滴加甲基丙烯酸酐具体是：在油浴锅中搅拌加热壳聚糖溶液至50℃，将浓度为94％甲基丙烯酸酐以0.5速度滴加于壳聚糖溶液中，用磁力搅拌器进行搅拌，转速为1000r/min，搅拌4h。需要说明的是，甲基丙烯酸酐质量体积可以根据S1中壳聚糖的质量体积以及S3中甲基丙烯酸酯化壳聚糖溶液的质量体积浓度推算出来的，此为本领域人员熟知过程，在此不再赘述。
S2中透析冻干具体是：在室温下，将混合物置于透析袋中进行蒸馏水渗析5d，渗析结束后，将混合物在-40℃温度下、5mbar压力下冷冻干燥24h。本实施例渗析所使用的透析袋为美国Sigma的分子量为12-14kDa的透析袋。
需要说明的是，壳聚糖溶液中滴加甲基丙烯酸酐溶液，如图1所示，壳聚糖会和甲基丙烯酸酐反应生成甲基丙烯酸酯化壳聚糖。壳聚糖完全反应后形成混合物应为甲基丙烯酸酯化壳聚糖和残留的甲基丙烯酸酐，所以使用渗析袋将 残留的甲基丙烯酸酐过滤掉获得纯品甲基丙烯酸酯化壳聚糖。
S3：制备甲基丙烯酸酯化壳聚糖溶液；
具体是，取甲基丙烯酸酯化壳聚糖纯品配制成5～50ml质量体积浓度为2％w/v的溶液，加入光引发剂后搅拌均匀，然后置于4℃下保存；本实施例中搅拌2h后甲基丙烯酸酯化壳聚糖溶液与引发剂混合均匀。需要说明的是，甲基丙烯酸酯化壳聚糖纯品的量可以根据甲基丙烯酸酯化壳聚糖溶液的质量体积浓度推算出来，此过程为本领域人员所熟知，在此不再赘述。。
S4：制备HA-DA-AEMA纯品；
具体是，分别称量4mmol的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、4mmol的N-羟基琥珀酰亚胺、2mmol的多巴胺和2mmol的甲基丙烯酸-2-氨基乙基酯酸置入避光离心管中；
将5～50ml质量体积比为1％w/v透明质酸钠溶液置于双口烧瓶中，然后使用0.1mol/L盐酸调节透明质酸钠溶液溶液PH值至5.0，然后加入称量好的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐并搅拌20min，然后依次加入称量好的N-羟基琥珀酰亚胺、多巴胺和甲基丙烯酸-2-氨基乙基酯酸获得混合溶液，透析冻干后获得HA-DA-AEMA纯品；本实施例中，调节PH的过程为：将PH仪的探头放入双口烧瓶，待PH仪读数稳定后，开始使用滴管向烧杯内滴加0.1mol/L盐酸，等到PH仪读数到5.0时，停止添加盐酸溶液。透明质酸钠、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N-羟基琥珀酰亚胺、多巴胺和甲基丙烯酸-2-氨基乙基酯酸合成HA-DA-AEMA的机理如图2所示。
S4中透析冻干具体是：在常温下，将承载混合溶液的容器置于磁力搅拌器上，调节搅拌器转速为850r/min，搅拌24h；
搅拌结束后，将混合溶液置于透析袋中，用PBS透析3天，透析结束后，将混合物在-80℃温度下、300mbar压力下冷冻干燥24h。
S4中所述透明质酸钠分子质量为9万，多巴胺分子质量为189.64，甲基丙烯酸-2-氨基乙基酯酸分子质量为165.62。
S5：制备HA-DA-AEMA溶液；
具体是，取HA-DA-AEMA纯品配制成5～50ml质量体积浓度为2％w/v的溶液，加入光引发剂后搅拌均匀，然后置于4℃下保存；
S6：将HA-DA-AEMA溶液和甲基丙烯酸酯化壳聚糖溶液分别固化成与组织粘附的内层生物凝胶和抗粘连的外层生物凝胶；
具体是，将HA-DA-AEMA溶液平铺皿上，加入光引发剂，用365nm紫外灯光照射30～90s；
待HA-DA-AEMA溶液从液态变为液-固态后将甲基丙烯酸酯化壳聚糖溶液平铺到HA-DA-AEMA液-固态上，加入光引发剂，然后用365nm紫外灯光照射30～150s，直至两种溶液全部变为固态，由与组织粘附的内层生物凝胶和抗粘连的外层生物凝胶构成的抗肠粘连双层生物凝胶形成。与组织粘附的内层生物凝胶和抗粘连的外层生物凝胶在受损肠道上构成的抗肠粘连双层生物凝胶的情况如图3所示。
本实施例中，光引发剂为Irgacure 2959或者LAP，Irgacure 2959和LAP质量体积分数都为0.1％w/v。
需要说明的是，本实施例中所使用的原料来源都没有特殊要求，采用本领域人员熟知的市售产品即可。
该抗肠粘连双层生物凝胶的制备方法，利用紫外光交联方式，使得由甲基丙烯酸酯化壳聚糖构成抗粘连外层生物凝胶和由HA-DA-AEMA构成与组织粘附的内层生物凝胶之间发生化学交联，紧密结合成整体，形成的双层生物凝胶既能起到固定肠道伤口的作用，防止炎性渗出和出血，又能避免粘性面和腹腔壁面或者肠道之间结合，防止不必要的粘连。
实施例2。
一种抗肠粘连双层生物凝胶的制备方法，其它特征与实施例1相同，不同之处在于：S1中预先制备壳聚糖溶液具体是：将质量体积分数为10％w/v的壳聚糖溶于浓度为5％醋酸中，用磁力搅拌器进行搅拌，转速为1000r/min，搅拌48h。
S1中所述壳聚糖的脱乙酰度范围为90％，分子质量为375000g·mol-1，粘 度为2000。
S2中滴加甲基丙烯酸酐具体是：在油浴锅中搅拌加热壳聚糖溶液至60℃，将浓度为95％甲基丙烯酸酐以1ml/min速度滴加于壳聚糖溶液中，用磁力搅拌器进行搅拌，转速为1000r/min，搅拌6h。
S3中甲基丙烯酸酯化壳聚糖溶液质量体积浓度为10％w/v。
S4中分别称量5mmol的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、5mmol的N-羟基琥珀酰亚胺、5mmol的多巴胺和3mmol的甲基丙烯酸-2-氨基乙基酯酸置入避光离心管中；
将质量体积比为20％w/v透明质酸钠溶液置于容器中，使用0.3mol/L盐酸调节透明质酸钠溶液溶液PH值至6.0，然后加入称量好的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐并搅拌30min，然后依次加入称量好的N-羟基琥珀酰亚胺、多巴胺和甲基丙烯酸-2-氨基乙基酯酸获得混合溶液，透析冻干后获得HA-DA-AEMA纯品；
S4中透析冻干具体是：在常温下，将承载混合溶液的容器置于磁力搅拌器上，调节搅拌器转速为1000r/min，搅拌48h；
搅拌结束后，将混合溶液置于透析袋中，用PBS透析5天，透析结束后，将混合物在-80℃温度下、400mbar压力下冷冻干燥48h。
S4中所述透明质酸钠分子质量为10万，多巴胺分子质量为189.64，甲基丙烯酸-2-氨基乙基酯酸分子质量为165.62。
S5中HA-DA-AEMA溶液质量体积浓度为10％w/v。
Irgacure 2959和LAP质量体积分数都为20％w/v。
该抗肠粘连双层生物凝胶的制备方法，利用紫外光交联方式，使得由甲基丙烯酸酯化壳聚糖构成抗粘连外层生物凝胶和由HA-DA-AEMA构成与组织粘附的内层生物凝胶之间发生化学交联，紧密结合成整体，形成的双层生物凝胶既能起到固定肠道伤口的作用，防止炎性渗出和出血，又能避免粘性面和腹腔壁面或者肠道之间结合，防止不必要的粘连。
实施例3。
一种抗肠粘连双层生物凝胶的制备方法，其它特征与实施例1相同，不同之处在于：S1中预先制备壳聚糖溶液具体是：将质量体积分数为5％w/v的壳聚糖溶于浓度为3％醋酸中，用磁力搅拌器进行搅拌，调节搅拌器转速为1000r/min，搅拌36h。
S1中所述壳聚糖的脱乙酰度范围为83％，分子质量为350000g·mol-1，粘度为1500。
S2中滴加甲基丙烯酸酐具体是：在油浴锅中搅拌加热壳聚糖溶液至55℃，将浓度为94.5％甲基丙烯酸酐以0.75ml/min速度滴加于壳聚糖溶液中，用磁力搅拌器进行搅拌，转速为1000r/min，搅拌5h。
S3中甲基丙烯酸酯化壳聚糖溶液质量体积浓度为6％w/v。
S4中分别称量4.5mmol的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、4.5mmol的N-羟基琥珀酰亚胺、3mmol的多巴胺和2.5mmol的甲基丙烯酸-2-氨基乙基酯酸置入避光离心管中；
将质量体积比为10％w/v透明质酸钠溶液置于容器中，使用0.2mol/L盐酸调节透明质酸钠溶液溶液PH值至5.5，然后加入称量好的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐并搅拌25min，然后依次加入称量好的N-羟基琥珀酰亚胺、多巴胺和甲基丙烯酸-2-氨基乙基酯酸获得混合溶液，透析冻干后获得HA-DA-AEMA纯品；
S4中透析冻干具体是：在常温下，将承载混合溶液的容器置于磁力搅拌器上，调节搅拌器转速为920r/min，搅拌36h；
搅拌结束后，将混合溶液置于透析袋中，用PBS透析4天，透析结束后，将混合物在-80℃温度下、350mbar压力下冷冻干燥36h。
S4中所述透明质酸钠分子质量为9.5万，多巴胺分子质量为189.64，甲基丙烯酸-2-氨基乙基酯酸分子质量为165.62。
S5中HA-DA-AEMA溶液质量体积浓度为6％w/v。
Irgacure 2959和LAP质量体积分数都为0.1～20％w/v。
该抗肠粘连双层生物凝胶的制备方法，利用紫外光交联方式，使得由甲基 丙烯酸酯化壳聚糖构成抗粘连外层生物凝胶和由HA-DA-AEMA构成与组织粘附的内层生物凝胶之间发生化学交联，紧密结合成整体，形成的双层生物凝胶既能起到固定肠道伤口的作用，防止炎性渗出和出血，又能避免粘性面和腹腔壁面或者肠道之间结合，防止不必要的粘连。
实施例4。
一种抗肠粘连双层生物凝胶的制备方法，其它特征与实施例1相同，不同之处在于：S3中甲基丙烯酸酯化壳聚糖溶液质量体积浓度为4～7％w/v，S4透明质酸钠溶液的质量体积比为7％w/v～15％w/v，S5中HA-DA-AEMA溶液质量体积浓度为4～7％w/v。该抗肠粘连双层生物凝胶的制备方法，利用紫外光交联方式，使得由甲基丙烯酸酯化壳聚糖构成抗粘连外层生物凝胶和由HA-DA-AEMA构成与组织粘附的内层生物凝胶之间发生化学交联，紧密结合成整体，形成的双层生物凝胶既能起到固定肠道伤口的作用，防止炎性渗出和出血，又能避免粘性面和腹腔壁面或者肠道之间结合，防止不必要的粘连。
实施例5。
一种抗肠粘连双层生物凝胶的制备方法，其它特征与实施例1相同，不同之处在于：S3中甲基丙烯酸酯化壳聚糖溶液质量体积浓度为5～76％w/v，S4透明质酸钠溶液的质量体积比为9％w/v～12％w/v，S5中HA-DA-AEMA溶液质量体积浓度为5～6％w/v。该抗肠粘连双层生物凝胶的制备方法，利用紫外光交联方式，使得由甲基丙烯酸酯化壳聚糖构成抗粘连外层生物凝胶和由HA-DA-AEMA构成与组织粘附的内层生物凝胶之间发生化学交联，紧密结合成整体，形成的双层生物凝胶既能起到固定肠道伤口的作用，防止炎性渗出和出血，又能避免粘性面和腹腔壁面或者肠道之间结合，防止不必要的粘连。
实施例6。
一种双层生物凝胶，采用抗肠粘连双层生物凝胶的制备方法制备。抗肠粘连双层生物凝胶的制备方法包括以下步骤：S1：预先制备壳聚糖溶液；S2：制备甲基丙烯酸酯化壳聚糖纯品；S3：制备甲基丙烯酸酯化壳聚糖溶液；S4：制 备HA-DA-AEMA纯品；S5：HA-DA-AEMA溶液制备；S6：将HA-DA-AEMA溶液和甲基丙烯酸酯化壳聚糖溶液分别固化成与组织粘附的内层生物凝胶和抗粘连的外层生物凝胶。
以腹腔手术后使用双层生物凝胶为例说明双层生物凝胶的使用方法：在腹腔手术结束但没有缝合伤口之前，将HA-DA-AEMA溶液涂布于手术切口、手术暴露肠道表面、肠胃吻合口等，用365nm紫外灯光照射30s另其由液态转化为液-固态形成与组织粘附的内层生物凝胶，然后将甲基丙烯酸酯化壳聚糖溶液涂布在与组织粘附的内层生物凝胶上后，用365nm紫外灯光照射60s，待HA-DA-AEMA溶液和甲基丙烯酸酯化壳聚糖溶液都凝固后，双层凝胶形成，缝合伤口。其中，甲基丙烯酸酯化壳聚糖溶液凝固成抗粘连的外层生物凝胶。
该双层生物凝胶，与组织粘附的内层生物凝胶可以和肠道表面紧密结合，能够起到固定肠道伤口的作用，防止炎性渗出和出血，抗粘连外层生物凝胶可以避免与组织粘附的内层生物凝胶粘性面和腹腔或者和其它肠道粘连，避免了肠粘连给患者带来的痛苦。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。

Claims (10)

  1. 一种抗肠粘连双层生物凝胶的制备方法，其特征在于，包括下列步骤：
    S1：预先制备壳聚糖溶液；
    S2：制备甲基丙烯酸酯化壳聚糖纯品；
    具体是，在壳聚糖醋酸溶液中滴加甲基丙烯酸酐溶液，将反应形成的混合物透析冻干获得甲基丙烯酸酯化壳聚糖纯品；
    S3：制备甲基丙烯酸酯化壳聚糖溶液；
    具体是，取甲基丙烯酸酯化壳聚糖纯品配制成5～50ml质量体积浓度为2～10％w/v的溶液，加入光引发剂后搅拌均匀，然后置于4℃下保存；
    S4：制备HA-DA-AEMA纯品；
    具体是，分别称量4～5mmol的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、4～5mmol的N-羟基琥珀酰亚胺、2～5mmol的多巴胺和2～3mmol的甲基丙烯酸-2-氨基乙基酯酸置入避光离心管中；
    将5～50ml质量体积比为1％w/v～20％w/v透明质酸钠溶液置于容器中，使用0.1～0.3mol/L盐酸调节透明质酸钠溶液溶液PH值至5.0～6.0，然后加入称量好的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐并搅拌20～30min，然后依次加入称量好的N-羟基琥珀酰亚胺、多巴胺和甲基丙烯酸-2-氨基乙基酯酸获得混合溶液，透析冻干后获得HA-DA-AEMA纯品；
    S5：制备HA-DA-AEMA溶液；
    具体是，取HA-DA-AEMA纯品配制成5～50ml质量体积浓度为2～10％w/v的溶液，加入光引发剂后搅拌均匀，然后置于4℃下保存；
    S6：将HA-DA-AEMA溶液和甲基丙烯酸酯化壳聚糖溶液分别固化成与组织粘附的内层生物凝胶和抗粘连的外层生物凝胶；
    具体是，将HA-DA-AEMA溶液平铺皿上，加入光引发剂，用365nm紫外灯光照射30～90s；
    待HA-DA-AEMA溶液从液态变为液-固态后将甲基丙烯酸酯化壳聚糖溶液 平铺到HA-DA-AEMA液-固态上，加入光引发剂，然后用365nm紫外灯光照射30～150s，直至两种溶液全部变为固态，由与组织粘附的内层生物凝胶和抗粘连的外层生物凝胶构成的抗肠粘连双层生物凝胶形成。
  2. 根据权利要求1所述的抗肠粘连双层生物凝胶的制备方法，其特征在于，S1中预先制备壳聚糖溶液具体是：将质量体积比为3％w/v～10％w/v的壳聚糖溶于50～200ml浓度为1％～5％醋酸中，用磁力搅拌器进行搅拌，调节搅拌器转速为1000r/min，搅拌24～48h。
  3. 根据权利要求1所述的抗肠粘连双层生物凝胶的制备方法，其特征在于，S3中甲基丙烯酸酯化壳聚糖溶液质量体积浓度为4～7％w/v，S4透明质酸钠溶液的质量体积比为7％w/v～15％w/v，S5中HA-DA-AEMA溶液质量体积浓度为4～7％w/v。
  4. 根据权利要求1所述的抗肠粘连双层生物凝胶的制备方法，其特征在于，S2中滴加甲基丙烯酸酐具体是：在油浴锅中搅拌加热壳聚糖溶液至50℃～60℃，将浓度为94％～95％甲基丙烯酸酐以0.5～1ml/min速度滴加于壳聚糖溶液中，用磁力搅拌器进行搅拌，转速为1000r/min，搅拌4～6h。
  5. 根据权利要求1所述的抗肠粘连双层生物凝胶的制备方法，其特征在于，S2中透析冻干具体是：在室温下，将混合物置于透析袋中进行蒸馏水渗析5～6d，渗析结束后，将混合物在-40℃～80℃温度下、5～100mbar压力下冷冻干燥24～48h。
  6. 根据权利要求1所述的抗肠粘连双层生物凝胶的制备方法，其特征在于，S4中透析冻干具体是：在常温下，将承载混合溶液的容器置于磁力搅拌器上，调节搅拌器转速为850～1000r/min，搅拌24～48h；
    搅拌结束后，将混合溶液置于透析袋中，用PBS透析3～5天，透析结束后，将混合物在-80℃温度下、300～400mbar压力下冷冻干燥24～48h。
  7. 根据权利要求1至6任意一项所述的抗肠粘连双层生物凝胶的制备方法，其特征在于，光引发剂为Irgacure 2959或者LAP，Irgacure 2959和LAP质量体积分数都为0.1～20％w/v。
  8. 根据权利要求1所述的抗肠粘连双层生物凝胶的制备方法，其特征在于，S1中所述壳聚糖的脱乙酰度范围为75～90％，分子质量为310000～375000g·mol -1，粘度为800-2000。
  9. 根据权利要求1所述的抗肠粘连双层生物凝胶的制备方法，其特征在于，S4中所述透明质酸钠分子质量为9万～10万，多巴胺分子质量为189.64，甲基丙烯酸-2-氨基乙基酯酸分子质量为165.62。
  10. 一种双层生物凝胶，其特征在于，采用权利要求1至9任意一项所述的抗肠粘连双层生物凝胶的制备方法制备。
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