WO2022262012A1 - 一种化学交联透明质酸水凝胶及其制备方法与应用 - Google Patents

一种化学交联透明质酸水凝胶及其制备方法与应用 Download PDF

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WO2022262012A1
WO2022262012A1 PCT/CN2021/103513 CN2021103513W WO2022262012A1 WO 2022262012 A1 WO2022262012 A1 WO 2022262012A1 CN 2021103513 W CN2021103513 W CN 2021103513W WO 2022262012 A1 WO2022262012 A1 WO 2022262012A1
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hyaluronic acid
cross
linking
lithium bromide
hydrogel
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郑兆柱
汪涛
王晓沁
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Suzhou University
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Suzhou University
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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
    • 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 chemically cross-linked hyaluronic acid hydrogel and its preparation method and application, belonging to the field of material technology.
  • tissue fillers Due to a series of problems such as defects and deformities of human tissues and organs caused by congenital or acquired reasons, coupled with the needs of the medical and aesthetic industry, researchers and medical device companies have been exploring a safe, convenient and reliable filler. To reshape and promote tissue repair, since the 1940s, due to the further development of material science, many implant materials have been used as tissue fillers for human tissue filling. In the past three decades, through the means of tissue filling The modality used to treat patients has increased in frequency, with the number of procedures involving soft tissue augmentation in the United States increasing from 1.6 million in 2011 to 2.4 million in 2015. Tissue filling is to achieve tissue filling by expanding volume. In recent decades, dermal fillers composed of a variety of high-viscosity liquids or polymer particle suspensions have been used to fill wrinkles and acne scars. The currently marketed tissue fillers are mainly collagen and hyaluronic acid.
  • Hyaluronic acid is a long-chain polymeric mucopolysaccharide widely distributed in various parts of the human body. It has good hydrophilicity, viscoelasticity, lubricity and biocompatibility, and has been widely used in the medical field. Natural hyaluronic acid has poor stability, is sensitive to hyaluronidase and free radicals, and has a short retention time in the body. ) to cross-link it, and regulate its in vivo retention time through the degree of cross-linking.
  • the cross-linking process is all under alkaline environment, preferably pH 11-13.
  • This method has low cross-linking efficiency and low reaction recovery rate (gel conversion rate), which will inevitably increase the injection frequency of the product and make the product expensive. Therefore, finding a high-efficiency cross-linked hydrogel and its preparation method is the key to solving the material demand and cost reduction in the field of tissue fillers.
  • the present invention provides a high-efficiency cross-linked hyaluronic acid, a preparation method and an application thereof.
  • the basic idea is that hyaluronic acid is a long-chain polysaccharide that has a higher degree of freedom in lithium bromide solution and exposes more reactive groups.
  • the chemical cross-linking reaction can significantly improve the reaction uniformity, cross-linking efficiency and product recovery rate by reacting in lithium bromide solution.
  • the present invention provides a chemically cross-linked hyaluronic acid hydrogel and a preparation method thereof.
  • the long-chain polysaccharide has a higher degree of freedom in the lithium bromide solution, exposes more reactive groups, and improves the reaction cross-linking efficiency and product recovery rate.
  • the first object of the present invention is to provide a chemical cross-linked hyaluronic acid hydrogel, said hyaluronic acid hydrogel is made of hyaluronic acid or its salt in lithium bromide solution, cross-linked by chemical cross-linking agent get.
  • the chemical crosslinking agent is diglycidyl ether, divinyl sulfone, 1,2,7,8-dioxoctane, 1,3-diepoxybutane and sodium trimetaphosphate one or more of two.
  • the molecular weight of the hyaluronic acid is 400k-3000kDa.
  • the molecular weight of the hyaluronic acid is preferably 500k-2500kDa, more preferably 1000k-2200kDa.
  • the second object of the present invention is to provide the preparation method of the chemically cross-linked hyaluronic acid hydrogel, comprising the following steps:
  • step S2 a step of removing lithium bromide, unreacted chemical cross-linking agent and unreacted hyaluronic acid after the reaction is also included.
  • the mass fraction of the hyaluronic acid or its salt is 1 mg/mL-300 mg/mL.
  • the mass fraction of the hyaluronic acid or its salt is preferably 10 mg/mL-250 mg/mL, more preferably 20 mg/mL-200 mg/mL.
  • the mass volume ratio of the hyaluronic acid or its salt to the chemical cross-linking agent is 1 g: 0.2-1000 ⁇ L.
  • the mass volume ratio of the hyaluronic acid or its salt to the chemical cross-linking agent is preferably 1 g: 2-800 ⁇ L, more preferably 1 g: 5-500 ⁇ L.
  • the lithium bromide concentration is 0.1-10M.
  • the lithium bromide concentration is preferably 1-10M, more preferably 2-9.8M.
  • cross-linking reaction is carried out at a reaction temperature of -80-80° C. for 3 minutes to 72 hours.
  • the third object of the present invention is to provide the application of the chemically cross-linked hyaluronic acid hydrogel, including the application as a filling or repairing material for soft tissues, hard tissues or cavities.
  • the fourth object of the present invention is to provide a hydrogel composition comprising the chemically cross-linked hyaluronic acid hydrogel.
  • the chemically cross-linked hyaluronic acid hydrogel also contains one or more combinations of moisturizing agents, bioactive agents, cosmetic active agents, cell adhesion agents, cells, extracellular matrix, and drugs.
  • Moisturizers include: glycerin, polyglutamic acid and the like.
  • the bioactive agents include: cell growth factors, peptides, peptidomimetics, antibodies, nucleic acids and polysaccharides.
  • the cells include: stem cells, adult cells, osteoblasts, chondrocytes, adipocytes, etc.
  • the cosmetic active agents include: anti-aging agents, anti-free radical agents, antioxidants, hydrating agents, whitening agents, coloring agents, sunscreen agents, muscle relaxants, etc.
  • the present invention adopts lithium bromide cross-linking instead of traditional alkaline reaction conditions, can reduce the amount of cross-linking agent, accelerate the speed of cross-linking reaction, improve product recovery (gel conversion rate), compared with the traditional preparation method, the prepared cross-linking agent for injection Linked hyaluronic acid gel products have higher cross-linking efficiency and product recovery rate (gel conversion rate), which significantly prolongs the maintenance time of the gel in vivo.
  • Fig. 1 is the reaction situation and reaction efficiency of hyaluronic acid in alkaline system (0.25M sodium hydroxide) and lithium bromide system.
  • A The reaction of hyaluronic acid in the sodium hydroxide and lithium bromide system;
  • B The reaction efficiency of hyaluronic acid in the sodium hydroxide and lithium bromide system.
  • Figure 2 shows the degree of modification of chemically cross-linked hyaluronic acid prepared by different reaction systems.
  • Figure 3 shows the in vitro degradation of chemically cross-linked hyaluronic acid prepared in different reaction systems.
  • the inventors tested the molecular weight of hyaluronic acid or its salt, the reaction mass fraction, the type of cross-linking agent, the amount of cross-linking agent added, the reaction temperature and the reaction time.
  • hyaluronic acid or its salts 400kDa, 500kDa, 700kDa, 900kDa, 1000kDa, 1300kDa, 1400kDa, 1500kDa, 1600kDa, 1800kDa, 2000kDa, 2100kDa, 2200kDa, 2400kDa, 2500kDa, 2700kDa, 2800kDa, 300
  • Reaction mass fraction 1mg/mL, 2mg/mL, 5mg/mL, 10mg/mL, 15mg/mL, 20mg/mL, 30mg/mL, 50mg/mL, 75mg/mL, 90mg/mL, 100mg/mL, 130mg/mL mL, 140mg/mL, 160mg/mL, 180mg/mL, 200mg/mL, 210mg/mL, 230mg/mL, 250mg/mL, 290mg/mL, 300mg/mL
  • crosslinking agents diglycidyl ether, divinyl sulfone, 1,2,7,8-dioxoctane, 1,3-bicyclic
  • Butane oxide sodium trimetaphosphate.
  • Amount of cross-linking agent added 1g: 0.2 ⁇ L, 1g: 0.40 ⁇ L, 1g: 0.7 ⁇ L, 1g: 1 ⁇ L, 1g: 4 ⁇ L, 1g: 7 ⁇ L, 1g: 8 ⁇ L, 1g: 10 ⁇ L, 1g: 30 ⁇ L, 1g: 60 ⁇ L, 1g : 80 ⁇ L, 1g: 100 ⁇ L, 1g: 300 ⁇ L, 1g: 600 ⁇ L, 1g: 900 ⁇ L, 1g: 1000 ⁇ L
  • Reaction temperature -80, -20, 4, 25, 37, 45, 60, 80
  • Reaction time 3 minutes, 5 minutes, 10 minutes, 30 minutes, 45 minutes, 60 minutes, 1 hour, 3 hours, 5 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours.
  • sample group of the present invention is better than the control group in the degree of modification test. It shows that the present invention adopts the lithium bromide reaction system to significantly improve the crosslinking efficiency.
  • hyaluronic acid molecular weight 1400KDa
  • BDDE Glycidyl ether
  • W 2 is cross-linked hyaluronic acid
  • W 1 is hyaluronic acid added to the reaction system before cross-linking.
  • Ideal hyaluronic acid hydrogels can be obtained when the molecular weight of hyaluronic acid is in the range of 400k-3000kDa. In the range of 500k-2500kDa, the performance of hyaluronic acid hydrogel is more excellent. In the range of 1000k-2200kDa, the performance of hyaluronic acid hydrogel is further excellent.
  • Ideal hyaluronic acid hydrogel can be obtained when the mass fraction of hyaluronic acid or its salt is in the range of 1 mg/mL to 300 mg/mL. In the range of 10mg/mL-250mg/mL, the performance of hyaluronic acid hydrogel is more excellent. In the range of 20mg/mL ⁇ 200mg/mL, the performance of hyaluronic acid hydrogel is further excellent.
  • Ideal hyaluronic acid hydrogel can be obtained when the mass volume ratio of hyaluronic acid or its salt to the chemical crosslinking agent is in the range of 1 g: 0.2-1000 ⁇ L. In the range of 1g: 2-800 ⁇ L, the performance of hyaluronic acid hydrogel is more excellent. In the range of 1g: 5-500 ⁇ L, the performance of hyaluronic acid hydrogel is further excellent.
  • the concentration of lithium bromide is in the range of 0.1-10M, ideal hyaluronic acid hydrogel can be obtained.
  • the performance of hyaluronic acid hydrogel is more excellent.
  • the performance of hyaluronic acid hydrogel is further excellent.
  • Ideal hyaluronic acid hydrogels can be obtained in the reaction temperature range of -80 to 80°C.
  • Ideal hyaluronic acid hydrogels can be obtained within the reaction time range of 3 minutes to 72 hours.
  • Embodiment 2 Hyaluronic acid modified degree test
  • Embodiment 3 in vitro degradation test
  • Example 1 Take an appropriate amount of 0.5g and 1g groups in Example 1, chemically cross-linked hyaluronic acid gel (containing about 8 mg of HA), put it in a vial, add 4 mL of self-made hyaluronic acid gelase (enzyme activity 60U/mL), Vortex to mix. Shake in a water bath at 42°C, sample 50 ⁇ L every 10 minutes and dilute appropriately, measure the absorbance at 232 nm, until the absorbance no longer changes, the degradation is considered complete, and the time taken for complete degradation is recorded. The test results are shown in Figure 3.
  • the cross-linked hyaluronic acid gel for injection prepared in the example has a relatively long time of degradation in vitro, while the cross-linked hyaluronic acid gel for injection prepared in the comparative example has a relatively short time of degradation in vitro, indicating that the The cross-linked hyaluronic acid gel for injection prepared by the method of the present invention has excellent resistance to enzymolysis, which shows that the obtained cross-linked hyaluronic acid gel for injection has high cross-linking efficiency.
  • Example 4 Preparation of injectable gel composition, biosafety evaluation and animal subcutaneous filling experiment
  • the pushing force is tested according to the test method in Appendix B of YY/T 0962-2014 Cross-linked Sodium Hyaluronate Gel for Plastic Surgery.
  • the pushing force of the hyaluronic acid composition prepared by the present invention is 5-30N.
  • Osmotic pressure is tested according to the freezing point depression in the fourth part 0632 osmolarity determination method of "Pharmacopoeia of the People's Republic of China” (2015 edition).
  • the particle size of the injectable gel system is 250-350 mOsmol.
  • Particle size is determined according to the color development in the photochromic emission method in the fourth part 0982 particle size and particle size distribution determination method of "Pharmacopoeia of the People's Republic of China” (2015 edition).
  • the particle size of the injectable gel system is 10-300 ⁇ m.
  • Rotational viscosity is measured according to the third-party rotational viscometer measurement method of the fourth part 0633 viscosity measurement method of "Pharmacopoeia of the People's Republic of China” (2015 edition).
  • the kinematic viscosity of the injectable gel system is 10-80 mm 2 /s.
  • pH value test according to the fourth part 0631pH value determination method of "Pharmacopoeia of the People's Republic of China” (2015 edition).
  • the pH of the injectable gel system is 6.0-8.0.
  • the pressure test is carried out according to the second method in the fourth part 0821 metal detection method of "Pharmacopoeia of the People's Republic of China” (2015 edition).
  • the heavy metal residue of the injectable gel system is ⁇ 10 ⁇ g/g.
  • Hemolysis test shall be carried out according to the method stipulated in GB/T16886.4-2003. ⁇ 5% of the injectable gel system.
  • the test shall be carried out according to the method specified in GB/T16886.10-2005. ⁇ Class I of the injectable gel system.
  • Yaman toxicity test is carried out according to the method specified in GB/T16886.11-2011.
  • the injectable gel system has no toxicity of Yaman.
  • Subcutaneous implantation experiment Choose 30 healthy rats of about 250 g, inject the sample group of the present invention on the left side of the back, and inject the control group on the right side, and the injection volume is 1 mL.
  • the injection volume is 1 mL.
  • the results of the experiment found that there were 30 rats in the three-month effective quantity sample group and 28 in the control group; 28 in the six-month effective quantity sample group and 15 in the control group.

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Abstract

一种化学交联透明质酸水凝胶及其制备方法与应用，所述透明质酸水凝胶是由透明质酸或其盐在溴化锂溶液中，通过化学交联剂交联得到。采用溴化锂交联替代传统碱性反应条件，可以降低交联剂用量，加快交联反应速度，提高交联效率，提高凝胶转换效率，与传统制备方法相比，所制备的注射用交联透明质酸凝胶产品具有更高的交联效率及凝胶转换效率，明显延长凝胶体内的维持时间。

Description

一种化学交联透明质酸水凝胶及其制备方法与应用 技术领域
本发明涉及一种化学交联透明质酸水凝胶及其制备方法与应用，属于材料技术领域。
背景技术
由于先天或后天的原因造成人体组织和器官的缺损、畸形等一系列问题，再加上医美行业的需求，研究者和医疗器械企业一直在探索一种安全、方便且可靠的填充物，用以整形和促进组织修复，在20世纪40年代起，由于材料科学的进一步发展，许多植入材料被用作组织填充剂应用于人体组织填充，在过去的三十年中，通过组织填充的手段治疗患者的方式使用频率越来越高，美国涉及软组织填充的手术数量从2011年160万例增加到2015年的240万例。组织填充是通过扩充体积来实现组织填充，近几十年来，多种高粘度液体或聚合物颗粒悬浮液组成的真皮填充剂被应用于皱纹和痤疮疤痕下填充。目前已上市组织填充剂主要以胶原和透明质酸为主。
透明质酸是一种高分子长链黏多糖，广泛分布于人体各部位，具有良好的亲水性、黏弹性、润滑性和生物相容性，并且已经在医学领域得到广泛的应用。天然透明质酸存在稳定性差、对透明质酸酶及自由基敏感、体内保留时间短，常用的方法是使用交联剂(二乙烯基砜(DVS)或者丁二醇二缩水甘油醚(BDDE))将其交联，通过交联度来调控其体内保留时间。
目前，已经报道的文献中，交联过程均是在碱性环境下，优选pH 11-13。此方法交联效率低，反应回收率低(凝胶转化率)，必然使得产品的注射频次增加、产品价格昂贵。因此，寻找一种高效交联水凝胶及其制备方法是解决目前组织填充剂领域材料需求和成本降低的关键。
为了解决上述问题，本发明提供了一种高效交联透明质酸、制备方法及其应用。基本构思是透明质酸是长链多糖在溴化锂溶液中具有更高的自由度，暴露更多的反应基团。化学交联反应在溴化锂溶液中反应可以明显提高反应均匀性、交联效率和产品回收率。
发明内容
为解决上述问题，本发明提供了一种化学交联透明质酸水凝胶及其制备方法。利用透明 质酸是长链多糖在溴化锂溶液中具有更高的自由度，暴露更多的反应基团，提高反应交联效率和产品回收率。
本发明的第一个目的是提供一种化学交联透明质酸水凝胶，所述的透明质酸水凝胶是由透明质酸或其盐在溴化锂溶液中，通过化学交联剂交联得到。
进一步地，所述的化学交联剂为二缩水甘油醚、二乙烯基砜、1,2,7,8-二环氧辛烷、1,3-二环氧丁烷和三偏磷酸钠中的一种或两种以上。
进一步地，所述的透明质酸的分子量为400k-3000kDa。
进一步地，所述的透明质酸的分子量优选为500k-2500kDa，进一步优选为1000k-2200kDa。
本发明的第二个目的是提供所述的化学交联透明质酸水凝胶的制备方法，包括如下步骤：
S1、将透明质酸或其盐溶于溴化锂溶液，得到透明质酸溴化锂溶液，并将化学交联剂加入到透明质酸溴化锂溶液中，或预先加入到溴化锂溶液中；
S2、进行交联反应，得到所述的化学交联透明质酸水凝胶。
进一步地，在S2步骤中，还包括反应后去除溴化锂、未反应的化学交联剂和未反应的透明质酸的步骤。
进一步地，所述的透明质酸或其盐的质量分数为1mg/mL～300mg/mL。
进一步地，所述的透明质酸或其盐的质量分数优选为10mg/mL～250mg/mL，进一步优选为20mg/mL～200mg/mL。
进一步地，所述的透明质酸或其盐与所述的化学交联剂的质量体积比为1g：0.2-1000μL。
进一步地，所述的透明质酸或其盐与所述的化学交联剂的质量体积比优选为1g：2-800μL，进一步优选为1g：5-500μL。
进一步地，所述的溴化锂浓度为0.1-10M。
进一步地，所述的溴化锂浓度优选为1-10M，进一步优选2-9.8M。
进一步地，所述的交联反应是在反应温度-80～80℃条件下反应3分钟～72小时。
本发明的第三个目的是提供所述的化学交联透明质酸水凝胶的应用，所述的应用包括在作为软组织、硬组织或者腔体的填充或修复材料中的应用。
本发明的第四个目的是提供一种水凝胶组合物，包括由所述的化学交联透明质酸水凝胶。
进一步地，所述的化学交联透明质酸水凝胶中还包含保湿剂、生物活性剂、美容活性剂、 细胞附着剂、细胞、细胞外基质、药物中的一种或多种组合。
其中保湿剂包括：甘油、聚谷氨酸等。
其中生物活性剂包括：细胞生长因子、肽、拟肽、抗体、核酸和多糖等。
其中细胞包括：干细胞、成体细胞、成骨细胞、成软骨细胞、成脂肪细胞等。
其中美容活性剂包括：抗衰老剂、抗自由基剂、抗氧化剂、水合剂、美白剂、着色剂、防晒剂、肌肉松弛剂等。
本发明的有益效果是：
本发明采用溴化锂交联替代传统碱性反应条件，可以降低交联剂用量，加快交联反应速度，提高产品回收率(凝胶转化率)，与传统制备方法相比，所制备的注射用交联透明质酸凝胶产品具有更高的交联效率及产品回收率(凝胶转换率)，明显延长凝胶体内的维持时间。
附图说明：
图1为透明质酸在碱性体系中(0.25M氢氧化钠)和溴化锂体系中反应情况及反应效率。A：透明质酸在氢氧化钠和溴化锂体系中反应情况；B：透明质酸在氢氧化钠和溴化锂体系中的反应效率。
图2为不同反应体系制备化学交联透明质酸修饰度。
图3为不同反应体系制备化学交联透明质酸体外降解情况。
具体实施方式
下面结合具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
实施例1：不通过反应体系制备交联透明质酸
为了验证本发明适用的范围及其反应条件，发明人对透明质酸或其盐分子量、反应质量分数、交联剂种类、交联剂添加量，反应温度和反应时间进行了测试。
透明质酸或其盐分子量：400kDa、500kDa、700kDa、900kDa、1000kDa、1300kDa、1400kDa、1500kDa、1600kDa、1800kDa、2000kDa、2100kDa、2200kDa、2400kDa、2500kDa、2700kDa、2800kDa、3000kDa。
反应质量分数：1mg/mL、2mg/mL、5mg/mL、10mg/mL、15mg/mL、20mg/mL、30mg/mL、50mg/mL、75mg/mL、90mg/mL、100mg/mL、130mg/mL、140mg/mL、160mg/mL、180mg/mL、200mg/mL、210mg/mL、230mg/mL、250mg/mL、290mg/mL、300mg/mL
交联剂种类：二缩水甘油醚、二乙烯基砜、1,2,7,8-二环氧辛烷、1,3-二环
氧丁烷、三偏磷酸钠。
交联剂添加量：1g：0.2μL、1g：0.40μL、1g：0.7μL、1g：1μL、1g：4μL、1g：7μL、1g：8μL、1g：10μL、1g：30μL、1g：60μL、1g：80μL、1g：100μL、1g：300μL、1g：600μL、1g：900μL、1g：1000μL
反应温度：-80、-20、4、25、37、45、60、80
反应时间：3分钟、5分钟、10分钟、30分钟、45分钟、60分钟、1小时、3小时、5小时、8小时、12小时、24小时、48小时、72小时。
对照组仅有反应体系为0.25M氢氧化钠溶液。
实验发现本发明样品组在修饰度测试中均优于对照组。说明本发明采用溴化锂反应体系使交联效率得到显著的提高。
发明人列举了将0.05g、0.1g、0.5g和1g透明质酸(分子量1400KDa)分别溶于10mL 0.25M NaOH或者9.3M溴化锂中，迅速向反应体系中加入500μL 1,4-丁二醇二缩水甘油醚(BDDE)，并搅拌均匀，置于60℃烘箱反应3小时，观察反应体系中是否有块状凝胶形成，收集块状凝胶，水洗、冻干称重，计算交联效率。
交联效率＝W 2/W 1*100％
其中，W 2为交联透明质酸，W 1为交联前加入到反应体系中的透明质酸。从图一中可以看出透明质酸反应浓度为0.5mg/mL时，两反应体系均没有成凝胶；反应浓度为1mg/mL时，溴化锂体系成凝胶，回收率约5％，而氢氧化钠体系没有成凝胶；反应浓度为5mg/mL和10mg/mL时两体系均成凝胶，但氢氧化钠体系回收率低于20％，而溴化锂体系超过60％。可见，在溴化锂体系中BDDE和透明质酸的反应效率远高于氢氧化钠体系。
研究发现：使用二缩水甘油醚、二乙烯基砜、1,2,7,8-二环氧辛烷、1,3-二环氧丁烷和三偏磷酸钠作为交联剂均能得到较为理想的玻尿酸水凝胶。
在透明质酸的分子量为400k-3000kDa范围内，均能得到较为理想的玻尿酸水凝胶。在500k-2500kDa范围内，玻尿酸水凝胶性能更加优异。在1000k-2200kDa范围内，玻尿酸水凝胶性能进一步优秀。
在透明质酸或其盐的质量分数为1mg/mL～300mg/mL范围内，均能得到较为理想的玻尿酸水凝胶。在10mg/mL～250mg/mL范围内，玻尿酸水凝胶性能更加优异。在20mg/mL～200mg/mL范围内，玻尿酸水凝胶性能进一步优秀。
在透明质酸或其盐与所述的化学交联剂的质量体积比为1g：0.2-1000μL范围内，均能得到较为理想的玻尿酸水凝胶。在1g：2-800μL范围内，玻尿酸水凝胶性能更加优异。在1g：5-500μL范围内，玻尿酸水凝胶性能进一步优秀。
在溴化锂浓度为0.1-10M范围内，均能得到较为理想的玻尿酸水凝胶。在1-10M范围内，玻尿酸水凝胶性能更加优异。在2-9.8M范围内，玻尿酸水凝胶性能进一步优秀。
在反应温度-80～80℃范围内，均能得到较为理想的玻尿酸水凝胶。
在反应时间3分钟～72小时范围内，均能得到较为理想的玻尿酸水凝胶。
实施例2：透明质酸修饰度测试
实施例1中0.5g和1g组，所得到的交联透明质酸凝胶上的9处位置的样品，每个样品1g左右，测定9个样品修饰度，并计算RSD值。修饰度测试条件：称取0.25g纯化后的实施例一的样品于5mL容量瓶中，用酶解液定容至5mL，42℃静置2h，灭活后静置备用。使用高效液相色谱测其修饰度，色谱柱：SuperdexTM 75，进样量：20μL，流速：0.5mL/min，柱温：35℃，流动相：20mmol/L，波长：232nm。在其他实验条件均一致的情况下，修饰度越高，说明交联效率越高。9个取样点的修饰度越接近，说明交联均匀性越高。通过图二结果可以看出，使用本发明方法所得到的交联透明质酸凝胶上的9个取样的修饰度几乎完全一致，而对比例中修饰度差异则比较大，说明该本发明方法使得交联均匀性得到有效保证。对比碱性条件下反应所得样品可以得出，本发明所得样品的修饰度远远高于对比组，说明本发明采用微波反应方法使交联效率得到显著的提高。
实施例3：体外降解测试
取适量实施例一中0.5g和1g组，化学交联透明质酸凝胶(约含HA 8mg)，置于西林瓶中，加入自制透明质酸凝胶酶(酶活60U/mL)4mL，涡旋混匀。42℃水浴振摇，每10min取样50μL并适当稀释，在232nm下测吸光度，直至吸光度不再变化，认为降解完全，记录完全降解所用时间。测试结果见图三。实施例制备得到的注射用交联透明质酸凝胶在体外降解的时间相对较长，而对比例所制备得到的注射用交联透明质酸凝胶在体外降解的时间相对较短，说明采用本发明所述的方法制备得到的注射用交联透明质酸凝胶具有优异的抗酶解性能，从而说明了所得到的注射用交联透明质酸凝胶交联效率高。
实施例4：可注射凝胶组合物制备、生物安全性评价及动物皮下填充实验
将10g大分子量透明质酸(分子量1400KDa)溶于100mL 9.3M溴化锂中，迅速向反应体系中加入5mL 1,4-丁二醇二缩水甘油醚(BDDE)，并搅拌均匀，置于60℃烘箱反应3小时，将凝胶切成约1cm 3颗粒，加磷酸盐缓冲液使凝胶块溶胀、透析、除去未反应的交联剂和溴化锂，控制透明质酸浓度为1.5％，凝胶pH值为6.0-8.0，过120目不锈钢筛网，得到 交联透明质酸钠凝胶。以0.25M氢氧化钠反应体系做对照。
取小分子量透明质酸(分子量80KDa)1.0g，加入到100mL 0.5％的甘油水(也可选用0.9％生理盐水、磷酸盐、氨基酸等等渗体系)溶液中，搅拌至溶解，经5μm滤芯过滤，得到pH为6.0-8.0的非交联透明质酸钠凝胶。将透明质酸钠凝胶和非交联透明质酸钠凝胶以质量比为1:10混合，混合均匀后，在100℃下进行热处理60分钟，得到可注射透明质酸盐凝胶体系。并进行如下测试：
1、推挤力，按照YY/T 0962-2014整形手术用交联透明质酸钠凝胶中附录B测试方法进行检测。得本发明所制备的透明质酸组合物推挤力为5-30N。
2、渗透压，按照《中华人民共和国药典》(2015版)第四部0632渗透压摩尔浓度测定法中的冰点下降发进行测试。所述可注射凝胶体系的粒径为250-350mOsmol。
3、粒径，按照《中华人民共和国药典》(2015版)第四部0982粒度和粒度分布测定法中光色she发中的显色发测定。所述可注射凝胶体系的粒径为10-300μm。
4、旋转粘度，按照《中华人民共和国药典》(2015版)第四部0633粘度测定法第三方转转粘度计测定法测定。所述可注射凝胶体系的运动粘度为10-80mm 2/s。
5、pH值，按照《中华人民共和国药典》(2015版)第四部0631pH值测定法进行测试。所述可注射凝胶体系的pH值为6.0-8.0。
6、重金属，按照《中华人民共和国药典》(2015版)第四部0821金属检测法中第二法进行试压。所述可注射凝胶体系的重金属残留≤10μg/g。
7、溶血实验，按照GB/T16886.4-2003规定的方法进行试验。所述可注射凝胶体系的≤5％。
8、细胞毒性实验，按照GB/T16886.5-2008规定的方法进行试验。所述可注射凝胶体系的≤I级。
9、皮肤致敏实验，按照GB/T16886.10-2005规定的方法进行试验。所述可注射凝胶体系的≤I级。
10、亚曼毒性实验，按照GB/T16886.11-2011规定的方法进行试验。所述可注射凝胶体系无亚曼毒性。
11、皮下植入实验。选250g左右健康大鼠30只，背部左侧注射本发明样品组，右侧注射对照组，注射量1mL。跟踪观察受试打住一周、两周、一个月、三个月、六个月效果。实验结果发现三个月有效数量样品组30只、对照组28只；六个月有效数量样品组28只、对照组15只。说明本发明方法制备的注射用交联透明质酸凝胶的安全性与传统交联方法均能满足注射美容填充手术的需求，而长期有效性结果显示，本发明方法制备的注射用交联透 明质酸凝胶在体内的维持时间远远高于传统交联方式制备的产品，是一种非常理想的组织填充剂。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。

Claims (11)

  1. 一种化学交联透明质酸水凝胶，其特征在于，所述的透明质酸水凝胶是由透明质酸或其盐在溴化锂溶液中，通过化学交联剂交联得到。
  2. 根据权利要求1所述的化学交联透明质酸水凝胶，其特征在于，所述的化学交联剂为二缩水甘油醚、二乙烯基砜、1,2,7,8-二环氧辛烷、1,3-二环氧丁烷和三偏磷酸钠中的一种或两种以上。
  3. 根据权利要求1所述的化学交联透明质酸水凝胶，其特征在于，所述的透明质酸的分子量为400k-3000kDa。
  4. 一种化学交联透明质酸水凝胶的制备方法，其特征在于，包括如下步骤：
    S1、将透明质酸或其盐溶于溴化锂溶液，得到透明质酸溴化锂溶液，并将化学交联剂加入到透明质酸溴化锂溶液中，或预先加入到溴化锂溶液中；
    S2、进行交联反应，得到所述的化学交联透明质酸水凝胶。
  5. 根据权利要求4所述的方法，其特征在于，所述的透明质酸或其盐的质量分数为1mg/mL～300mg/mL。
  6. 根据权利要求4所述的方法，其特征在于，所述的透明质酸或其盐与所述的化学交联剂的质量体积比为1g：0.2-1000μL。
  7. 根据权利要求4所述的方法，其特征在于，所述的溴化锂浓度为0.1-10M。
  8. 根据权利要求4所述的方法，其特征在于，所述的交联反应是在反应温度-80～80℃条件下反应3分钟～72小时。
  9. 权利要求1～3任一项所述的化学交联透明质酸水凝胶的应用，其特征在于，所述的应用包括在作为软组织、硬组织或者腔体的填充或修复材料中的应用。
  10. 一种水凝胶组合物，其特征在于，包括由权利要求1～3任一项所述的化学交联透明质酸水凝胶。
  11. 根据权利要求10所述的水凝胶组合物，其特征在于，所述的化学交联透明质酸水凝胶中还包含保湿剂、生物活性剂、美容活性剂、细胞附着剂、细胞、细胞外基质、药物中的一种或多种组合。
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