WO2019033596A1 - 制备单相修饰透明质酸钠凝胶的方法 - Google Patents

制备单相修饰透明质酸钠凝胶的方法 Download PDF

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WO2019033596A1
WO2019033596A1 PCT/CN2017/111286 CN2017111286W WO2019033596A1 WO 2019033596 A1 WO2019033596 A1 WO 2019033596A1 CN 2017111286 W CN2017111286 W CN 2017111286W WO 2019033596 A1 WO2019033596 A1 WO 2019033596A1
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sodium hyaluronate
gel
added
daltons
phase
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PCT/CN2017/111286
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French (fr)
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冯夕江
陈燕
孙伟庆
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杭州协合医疗用品有限公司
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Priority to US16/022,788 priority Critical patent/US20190055368A1/en
Publication of WO2019033596A1 publication Critical patent/WO2019033596A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • 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
    • A61L2400/06—Flowable or injectable implant compositions
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
    • C08J2305/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof

Definitions

  • the invention relates to a preparation method and a product obtained by single-phase modified sodium hyaluronate gel, which is suitable for filling and repairing moderate to severe facial wrinkles or wrinkles, and belongs to the field of implanted medical beauty.
  • Medical beauty is a series of treatments that are carried out by means of medical means, including instruments, drugs, instruments and surgery, in order to achieve changes in the external form, color and partial improvement of physiological functions of the human body, and to enhance the aesthetics of the human body.
  • Minimally invasive injection plastic surgery develops rapidly, with minimal damage to normal tissues, minimal inflammation, swelling, minimal blood stasis, minimal complications, and minimal scarring.
  • the treatment time is short, the patient suffers little pain, the postoperative recovery is fast, the treatment effect is good, the safety is high, and the patient does not need to be hospitalized.
  • the health and natural properties of hyaluronic acid are particularly prominent.
  • Hyaluronic acid was first isolated from the vitreous of the bovine eye. It is widely found in the extracellular matrix of connective tissue. It is passed from D-glucuronic acid and N-acetylglucosamine. The ⁇ 3 bond and the ⁇ 1 ⁇ 4 bond alternately form a disaccharide unit, and then a linear polysaccharide composed of repeating disaccharide units.
  • Hyaluronic acid has no species and tissue specificity, has good histocompatibility, and the body rarely produces an immune response to it.
  • Hyaluronic acid is highly hydrophilic. This physicochemical property keeps hyaluronic acid gel-like even at very low concentrations.
  • Hyaluronic acid absorbs water and increases in volume, causing expansion pressure around it to support the surrounding. organization.
  • natural hyaluronic acid has a half-life of only 1 to 2 days in tissues and is decomposed into CO 2 and H 2 O by the hyaluronidase or oxygen free radical in the liver.
  • Hyaluronic acid also has the property of isovolumetric degradation, that is, when a part of hyaluronic acid is degraded, the remaining molecules can absorb more water to maintain the total volume until all the molecules are completely degraded.
  • Hyaluronic acid content decreases with age, which directly leads to the loss of moisture in the skin and the formation of wrinkles.
  • hyaluronic acid is clinically applied to skin rejuvenation treatment such as improving wrinkles and increasing tissue volume. Therefore, in order to become an ideal skin filling material, it is necessary to modify and crosslink it, so that a product with a more stable molecular structure and a longer retention time can be obtained. Because of the lack of antigen specificity, hyaluronic acid rarely occurs in different species or tissues, and it has a good conformational stiffness and a strong water-locking function. Due to its excellent biocompatibility and filling effect, it is gradually replacing collagen and becoming the current mainstream skin filling material.
  • the object of the present invention is to provide a method for preparing a single-phase sodium hyaluronate gel, which has good biocompatibility and excellent enzymatic hydrolysis resistance, and the method is convenient to operate and easy to realize the production of industrial medical equipment.
  • the present invention includes the following steps:
  • a method of preparing a single phase modified sodium hyaluronate gel characterized in that it comprises the following steps:
  • the molar ratio of the crosslinking agent to the sodium hyaluronate is 9% to 15%, and rapidly mixing at 20 to 40 minutes to form a gel;
  • the polymer is of natural origin, using a polymer of natural origin for better biocompatibility, that is to say, causing less risk of inflammatory reactions, sodium hyaluronate as described in step (1) Sodium hyaluronate produced by bacterial fermentation is used.
  • the alkaline condition described in the step (1) is preferably potassium hydroxide or sodium hydroxide, and the pH is preferably 13-14.
  • the crosslinking agent in the step (2) is a biofunctional molecule selected from the group consisting of an epoxy compound, a halogenated alcohol, and a divinyl sulfone compound.
  • Preferred epoxides are compounds selected from the group consisting of 1,4-butanediol diglycidyl ether (also known as 1,4-bis(2,3-epoxypropoxy)butane), 1 ⁇ ( 2,3-epoxypropyl) 2,3-epoxycyclohexane and 1,2-ethanediol diglycidyl ether.
  • the constant temperature water bath described in the step (3) has a temperature of 27 to 60 ° C, preferably 30 to 50 ° C.
  • the dialysis described in the step (4) employs a dialysis membrane having a molecular weight of 20,000 Daltons, preferably 15,000 Daltons.
  • the anesthetic agent is added to the step (5) and then homogenized; the anesthetic agent is preferably lidocaine hydrochloride; the mass of the anesthetic is 0.1% to 0.5%, preferably 0.2% to 0.4%.
  • the mobile phase described in the step (6) is the same as the sodium hyaluronate raw material of the cross-linking reaction, and is derived from the bacterial fermentation method, has the same molecular weight, and is consistent with the gel content, and the mobile phase accounts for the total mass ratio of 5 % to 50%, preferably 10% to 30%.
  • the hyaluronic acid having a molecular weight of 2 million Daltons is dissolved in a lye of pH 14 and 12% of the crosslinking agent 1,4 - butanediol diglycidyl ether is added, and the mixture is quickly mixed for 30 minutes.
  • the mixture was cross-linked in a water bath at 40 ° C, dialyzed against a dialysis membrane which precipitated 15,000 daltons, and 0.2% to 0.4% of lidocaine hydrochloride was added thereto, and 20% of the mobile phase was added to prepare a single-phase modified sodium hyaluronate gel.
  • the prepared single-phase sodium hyaluronate gel is stored in a vial or a prefilled syringe and stored in a sealed state after moist heat sterilization.
  • the gel of the invention is preferably injected.
  • the present invention provides a biocompatible single phase crosslinked gel which effectively avoids the disadvantages of the presence of a biphasic gel in the prior art, which at the same time has the advantage of being easy to use and longer lasting in clinical applications.
  • the invention starts from the research on the degree of hyaluronic acid modification of the human body, and controls the pre-parameter parameters, adopts the unsaturated cross-linking method, and further homogenizes to obtain single-phase, multi-polymer, high-viscosity, injectable and long.
  • a continuous biocompatible sodium hyaluronate gel The biocompatibility and physical and chemical properties of the product are greatly improved.
  • the anesthetic drug lidocaine hydrochloride was added during the production process. The whole process is simple and easy to implement, and the result is stable and reliable.
  • the single phase modified sodium hyaluronate gel prepared according to the above process is capable of filling, separating or replacing biological tissue or increasing the volume of the tissue, or supplementing or replacing biological fluids.
  • Preferably used to fill, separate or replace biological tissue or increase the volume of the tissue for example, as a material for therapeutic applications, increase the volume of the vocal cords, increase the volume of the esophagus, urethral sphincter or other organs, etc., or for cosmetic purposes Wrinkles, covering scars or lips.
  • the gel constitutes a matrix comprising at least one dispersed active host. The gel is then used as a carrier that allows for the gradual release of the active subject from the liquid or biological tissue into which it is injected.
  • the single-phase gel is distinguished from the conventional two-phase gel, observed under the microstructure, and the conventional two-phase gel with particle size difference is shown in Fig. 1, and the single-phase homogeneous gel is shown in Fig. 2.
  • the two-phase contains solid phase particles and liquid phase.
  • the single-phase gel is a very stable colloidal phase, which is very similar to the degree of hyaluronic acid self-existing in the human body.
  • the structure is stable, the tissue affinity is extremely high, the shaping effect is more natural, and the lifting effect is remarkable, which effectively improves the unevenness of the skin surface after surgery, and is soft and elastic.
  • the high viscosity of the gel means that the gel is strongly recombined, rather than being unfolded or separated.
  • the corresponding excellent shear viscosity can effectively resist the shearing force generated after the injection, thereby reducing the diffusion and movement, and resisting the deformation caused by the external force. more stable. Therefore, the high viscosity cohesiveness of the gel contributes to high compatibility and long-term persistence in the body.
  • the high-viscosity characteristics of the single-phase gel of the present invention are not isolated, meaning that the absolute value of the viscosity of the gel in a specific test case is high, but the comprehensive evaluation of the elastic properties is combined with the industry.
  • the gel of the present invention generally has a larger phase angle than the two-phase gel, and the viscosity property is more outstanding.
  • the single-phase performance of the sodium hyaluronate gel prepared by the invention is outstanding, and the risk of inflammatory reaction and the appearance of granuloma are more and less reduced. Prolonged maintenance in the body provides the possibility of longer interval medical interventions, thereby improving the quality of life of patients.
  • the modified sodium hyaluronate gel prepared by the present invention is more injectable and longer lasting than other gels having the same degree of crosslinking.
  • Fig. 1 is a view showing the results of observation of a two-phase particle gel prepared by a conventional method under a microscope.
  • Fig. 2 is a view showing the results of observation of a single-phase homogeneous gel prepared by the method of the present invention under a microscope.
  • Figure 3 is a comparison of the enzymatic hydrolysis curves of Example 1, Example 3 and control biphasic gel.
  • Example 1 The gel of Example 1 was dialyzed into a dialysis membrane bag having a dialysis molecular weight of 15,000 Daltons to remove unreacted cross-linking agent and excess hydroxide ions, and then 0.3% of a 0.2 ⁇ m microporous membrane was added. Filtered lidocaine hydrochloride, adjust pH to neutral, homogenize. 10.06 g of mobile phase was added and thoroughly mixed to obtain a homogeneous single phase gel.
  • the gel was packaged into a prefilled syringe and sterilized by damp heat at 121 °C for 30 minutes.
  • the gel of the above example was tested for elasticity and viscosity using a rotary rheometer.
  • the test method was a dynamic frequency sweep with a test temperature of 25 ° C and a frequency range of 0.05 to 10 Hz.
  • the values of the elastic modulus (G') and the viscous modulus (G") at 1 Hz were compared.
  • the in vitro anti-enzymatic properties of the gel can indirectly reflect its maintenance time in the human body.
  • the present invention was subjected to an enzymatic hydrolysis test at a concentration of 7 U/ml of hyaluronidase using Examples 1 and 3 and a control commercially available two-phase gel. The results are shown in the following table, and the enzymatic curve is shown in Fig. 3.

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Abstract

本发明涉及一种制备单相修饰透明质酸钠凝胶的方法，该凝胶是生物相容性的，其通过合理的参数进行交联反应，并通过透析除去交联剂和交联环境以终止交联反应，均质化后与流动相的混合进一步增强凝胶的集聚性和可应用性，麻醉剂的添加是基于应用舒适性考虑，本发明还公开了凝胶及其用途。

Description

制备单相修饰透明质酸钠凝胶的方法 技术领域
本发明涉及一种单相修饰透明质酸钠凝胶的制备方法及所得产品，该产品适用于中重度面部皱纹或褶皱的填充修复，属于植入医学美容领域。
背景技术
医学美容通过医学手段，使用包括器械、药物、仪器及手术等，以达到改变人体外部形态、色泽及部分改善其生理功能，增强人体外在美感为目的，而进行的系列的治疗。微创注射植入整形美容发展迅速，其对正常的组织损伤最小，炎症反应最轻，肿胀、瘀血最轻，并发症最少，瘢痕最小。而且治疗时间短，患者痛苦小，术后恢复快，治疗效果好，安全性高，患者无需住院等。在注射类项目中透明质酸的健康、自然属性尤为突出，由于注射成本较低，操作时间较短，注射过程不会产生明显痛觉，不会对机体功能造成影响，因此不需要休要时间，易被消费者接受，其受欢迎程度日渐提升，注射透明质酸美容渐成趋势。
透明质酸(Hyaluronic acid、简称HA)最早是从牛眼玻璃体中首次分离出的，它广泛存在于人体结缔组织的细胞外基质中，由D‐葡萄糖醛酸与N‐乙酰葡糖胺通过β1‐3键及β1‐4键交替组成双糖单位，再由双糖单位重复组成的线性多糖。透明质酸没有种属和组织特异性，具有良好的组织相容性，机体很少对其产生免疫反应。透明质酸具有高度亲水性，这种理化性质使透明质酸即使在很低的浓度下，依然保持凝胶状，透明质酸吸水后体积增大，向周围产生膨胀压力使其可以支撑周围组织。然而天然透明质酸在组织中的半衰期仅为1～2天，会被透明质酸酶或氧自由基在肝脏中分解为CO2和H2O。透明质酸还具有等容降解的特性，即当一部分透明质酸降解时，剩下的分子可以吸收更多的水分以维持总体积的不变，直至所有的分子完全降解。透明质酸含量随着年龄的增长不断减少， 这就直接导致皮肤中水分的丢失进而皱纹形成。因而在临床上将透明质酸应用于改善皱纹及增加组织容积等皮肤年轻化治疗。所以，想要成为理想的皮肤填充材料就必须对其进行修饰交联，这样可以获得分子结构更加稳定，保持时间更长的产品。在不同的物种或组织间，透明质酸因为没有抗原特异性，所以它很少会发生过敏反应，同时具有良好的构象刚度和极强的锁水功能。由于其出色的生物相容性和填充效果，正逐步取代胶原蛋白，成为当前主流的皮肤填充材料。
当前市面上出现较多的是利用足量交联剂对透明质酸进行充分修饰，人为过筛，得到刚性强度比原生透明质酸大得多的固体颗粒状胶块，形成双相凝胶，在临床上因不同机体状况而屡有出现红肿疼痛、炎症、异物感和迟发性过敏等症状。
发明内容
本发明的目的是提供一种制备单相透明质酸钠凝胶方法，所得产品生物相容性好，耐酶解性能优异，同时该方法操作方便，易实现工业化医械产品的生产。为实现上述目的，本发明包括以下步骤：
制备单相修饰透明质酸钠凝胶的方法，其特征在于，它包括以下步骤：
(1)碱性条件pH值11‐‐14下配制浓度为质量百分比5％～15％的透明质酸钠溶液，透明质酸钠的分子量为150万～400万道尔顿；
(2)往步骤(1)的溶液中加入交联剂，交联剂与透明质酸钠的摩尔量比例为9％～15％，20‐‐40分钟快速混匀，形成凝胶；
(3)恒温水浴后静置；
(4)采用透析膜透析，除去未反应交联剂及氢氧根离子；
(5)均质化；
(6)加入流动相，充分混匀，获得高粘稳定化的单相修饰透明质酸钠凝胶。
优选地，聚合物是天然来源的，使用天然来源的聚合物具更好的生物相容性，也就是说用此引起较少的炎症反应风险，步骤(1)中所述的透明质酸钠选用细菌发酵法生产的透明质酸钠。
进一步地，步骤(1)中所述的碱性条件，优选氢氧化钾或氢氧化钠，pH值优选13‐‐14。
进一步地，步骤(2)中交联剂是选自环氧化物，卤代醇和二乙烯基砜的化合物的生物多功能分子。优选的环氧化物是选自以下的化合物：1,4‐丁二醇二缩水甘油醚(也称为1,4‐双(2,3‐环氧丙氧基)丁烷)，1‐(2,3‐环氧丙基)2,3‐环氧环己烷和1,2‐乙二醇二环氧甘油醚。
进一步地，步骤(3)中所述的恒温水浴，温度为27～60℃，优选为30～50℃。
进一步地，步骤(4)中所述的透析，采用析出分子量为20000道尔顿的透析膜，优选为15000道尔顿。
进一步地，步骤(5)中加入麻醉剂后再均质化；所述的麻醉剂优选采用盐酸利多卡因；麻醉剂质量含量为0.1％～0.5％，优选为0.2％～0.4％。
进一步地，步骤(6)中所述的流动相，其为与交联反应的透明质酸钠原料相同，源自细菌发酵法，分子量相同，与凝胶含量一致，流动相占总质量比例5％～50％，优选为10％～30％。
进一步地，在于优选分子量200万道尔顿的透明质酸溶于pH值14的碱液中，加入12％的交联剂1,4‐丁二醇二缩水甘油醚，30分钟快速混匀，40℃下水浴交联，用析出15000道尔顿的透析膜透析，加入0.2％～0.4％盐酸利多卡因，加入20％流动相，制得的单相修饰透明质酸钠凝胶。
进一步地，所制得的单相透明质酸钠凝胶经湿热灭菌后贮存于西林瓶或预灌封注射器中密封保存。
本发明凝胶优选注射。
本发明提出一种生物相容性的单相交联凝胶，有效避免了背景技术中双相凝胶存在的缺点，其同时具有在临床应用中容易使用和更长持续时间的优点。
本发明从研究接近人体自身透明质酸修饰程度入手，通过控制前期参数，采用非饱和交联方法，并进一步均质化，得到具有单相，多聚，高粘，可注射和长 持续性的生物相容性透明质酸钠凝胶。产品的生物相容性和理化特性相比较有极大的改善。并且，为增加临床使用的舒适体验，制作过程中，添加有麻醉药盐酸利多卡因。整个过程操作简单易实现，结果稳定可靠。
根据上述工艺所制得的单相修饰透明质酸钠凝胶能够填充、分离或替换生物组织或增加所述组织的体积，或者补充或替代生物流体。优选地用于填充，分离或替换生物组织或增加所述组织的体积，例如，作为治疗应用的材料，增加声带的体积，增加食道、尿道括约肌或其他器官的体积等，或用于美容目的填充皱纹，掩盖疤痕或丰唇。优选地，凝胶构成包含至少一种分散的活性主体的基质。然后将凝胶用作允许从其注射的液体或生物组织中逐步释放所述活性主体的载体。
单相凝胶区别于常规的双相凝胶，显微结构下观察，常规以粒径区别的双相凝胶如图1，单相均质凝胶如图2。
双相含有固相颗粒和液相，应用时导致局部突兀不平滑，体内吸收降解先后不一，而单相凝胶是非常稳定的胶体相，与人体内自身存在的透明质酸修饰程度极为相似，结构稳定，组织亲和力极高，塑形效果更自然、提升效果显著，有效改善术后皮肤表面凹凸不平现象，柔和而富有弹性。凝胶高粘意味着凝胶的强烈重新组合成型趋向，而不是展开或分开，相应的优秀剪切粘度能够有效抵抗注入后产生的剪切力，从而减少扩散和移动，抵抗外力引起的变形，更加稳定。因此，凝胶高粘凝聚性有助于在体内获得高相容性和长时间持续性。
需特别说明的是，本发明单相凝胶的高粘特性，并不是孤立的意味着凝胶在特定测试情况下粘性的绝对值就高，而是要结合其弹性性能综合的评价，行业上有衡量凝胶流变性能的相角α，tgα即粘性与弹性的比值，本发明凝胶普遍具有比双相凝胶更大的相角，粘性性能更突出优异。
本发明制备的透明质酸钠凝胶单相性能突出，愈加减少炎症反应的风险和肉芽肿的出现。体内维持时间延长，为更长间隔医疗干预提供可能，从而提高患者生活质量。
本发明制备的修饰透明质酸钠凝胶，其比交联程度相同的其它凝胶在体内更具注射性，且持久性更长。
附图说明
图1为采用现有方法制成的双相颗粒凝胶在显微镜下观测的结果图。
图2为采用本发明方法制成的单相均质凝胶在显微镜下观测的结果图。
图3为实施例1、例3和对照双相凝胶的酶解曲线对比图。
具体实施方式
以下实施例仅以说明性示例的方式给出以说明本发明，帮助理解本发明，但是决不是限制本发明。
实施例一
称量2.02g分子量为160万道尔顿的透明质酸钠于烧杯中，加入15.05g 1％的氢氧化钠溶液，充分溶解。交联步骤在碱性介质中进行，容易形成非常牢固的醚键。加入60μl二乙烯基砜，彻底混匀，然后在50℃下反应4小时，静置过夜，获得交联凝胶。
实施例二
将实施例一的凝胶装入透析分子量为15000道尔顿的透析膜袋中透析，以除去未反应的交联剂和多余的氢氧根离子，然后加入0.3％经0.2μm微孔滤膜过滤的盐酸利多卡因，调节pH至中性，均质化。加入10.06g流动相，充分混匀，得到均质单相凝胶。
将凝胶封装入预灌封注射器，121℃湿热灭菌30分钟。
取产品于微纳99D颗粒图像仪下观察，得如附图2结果。
实施例三
称量5.03g分子量为230万道尔顿的透明质酸钠于烧杯中，加入55.2g 1％的氢氧化钠溶液，充分溶解。加入321μl1,4‐丁二醇二缩水甘油醚，彻底混匀，然后在40℃下反应4小时，静置过夜获得交联凝胶。然后将凝胶装入透析膜袋中透析，以除去未反应的交联剂和多余的氢氧根离子，再加入0.3％经0.2μm微孔 滤膜过滤的盐酸利多卡因，调节pH至中性，均质化。加入52.1g流动相，充分混匀，得到均质单相凝胶。
实施例四
称量10.02g分子量为190万道尔顿的透明质酸钠于烧杯中，加入160.1g 1％的氢氧化钠溶液，充分溶解。加入550μl 1,4‐丁二醇二缩水甘油醚，彻底混匀，然后在40℃下反应4小时，静置过夜获得交联凝胶。然后将凝胶放入磷酸缓冲盐中透析，以除去未反应的交联剂和多余的氢氧根离子，再加入0.3％经0.2μm微孔滤膜过滤的盐酸利多卡因，调节pH至中性，均质化。加入105.0g流动相，充分混匀，得到均质单相凝胶。
采用旋转流变仪对上述实施例凝胶进行弹性和粘性测试，测试方法为动态频率扫描，测试温度为25℃，频率变化范围为0.05～10Hz。比较1Hz下弹性模量(G’)和粘性模量(G”)的值。
对实施例每组样品取5个平行样，安装上注射针。推挤注射器排除前端少量空气至针尖部位出现凝胶小滴。将注射器置于拉力机上，以20mm/min的速度推动注射柄，记录压力，并计算其平均值。
本发明单相凝胶测试结果，并与当前市售某双相凝胶比较，如下表：
Figure PCTCN2017111286-appb-000001
凝胶的体外抗酶解性能能间接反映其在人体内的维持作用时间。本发明取实施例1和例3及对照市售双相凝胶在7U/ml的透明质酸酶浓度下进行酶解试验，结果如下表，酶解曲线见图3。
酶解率(％)
时间(h) 1 3 5 7 9
例1 29 49 72 80 89
例3 38 56 70 86 92
对照 64 88 93 100 100

Claims (10)

  1. 制备单相修饰透明质酸钠凝胶的方法，其特征在于，它包括以下步骤：
    (1)碱性条件pH值11‐‐14下配制浓度为质量百分比5％～15％的透明质酸钠溶液，透明质酸钠的分子量为150万～400万道尔顿；
    (2)往步骤(1)的溶液中加入交联剂，交联剂与透明质酸钠的摩尔量比例为9％～15％，20‐‐40分钟快速混匀，形成凝胶；
    (3)恒温水浴后静置；
    (4)采用透析膜透析，除去未反应交联剂及氢氧根离子；
    (5)均质化；
    (6)加入流动相，充分混匀，获得高粘稳定化的单相修饰透明质酸钠凝胶。
  2. 根据权利要求1所述的方法，其特征在于，步骤(1)中所述的透明质酸钠选用细菌发酵法生产的透明质酸钠。
  3. 根据权利要求1所述的方法，其特征在于，步骤(1)中所述的碱性条件，优选氢氧化钾或氢氧化钠，pH值优选13‐‐14。
  4. 根据权利要求1所述的方法，其特征在于，步骤(2)中所述的交联剂选自环氧化物，卤代醇和二乙烯基砜；优选的环氧化物是选自以下的化合物：1,4‐丁二醇二缩水甘油醚、1‐(2,3‐环氧丙基)2,3‐环氧环己烷和1,2‐乙二醇二环氧甘油醚。
  5. 根据权利要求1所述的方法，其特征在于，步骤(3)中所述的恒温水浴，温度为27～60℃，优选为30～50℃。
  6. 根据权利要求1所述的方法，其特征在于，步骤(4)中所述的透析，采用析出分子量为20000道尔顿的透析膜，优选为15000道尔顿。
  7. 根据权利要求1所述的方法，其特征在于，步骤(5)中加入麻醉剂后再均质化；所述的麻醉剂优选采用盐酸利多卡因；麻醉剂质量含量为0.1％～0.5％，优选为0.2％～0.4％。
  8. 根据权利要求1所述的方法，其特征在于，步骤(6)中所述的流动相，其为与交联反应的透明质酸钠原料相同，源自细菌发酵法，分子量相同，与凝胶含量一致，流动相占总质量比例5％～50％，优选为10％～30％。
  9. 根据权利要求1至8中的任意一项方法，其特征在于优选分子量200万道尔顿的透明质酸溶于pH值14的碱液中，加入12％的交联剂1,4-丁二醇二缩水甘油醚，30分钟快速混匀，40℃下水浴交联，用析出15000道尔顿的透析膜透析，加入0.2％～0.4％盐酸利多卡因，加入20％流动相，制得的单相修饰透明质酸钠凝胶。
  10. 根据权利要求1至9中的任意一项工艺所制得的单相修饰透明质酸钠凝胶在医学美容填充、替换或分离生物组织或增加所述组织的体积，或者补充或替代生物材料的应用。
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