CN116099038A

Movatterモバイル変換

Info

Publication number: CN116099038A
Application number: CN202310177716.8A
Authority: CN
Inventors: 刘艳秋; 郭丽伟; 张婷玥; 李若兰; 邵将
Original assignee: Southwest Jiaotong University
Current assignee: Southwest Jiaotong University
Priority date: 2023-02-27
Filing date: 2023-02-27
Publication date: 2023-05-12
Anticipated expiration: 2043-02-27

Abstract

The invention provides a bioactive hydrogel and a preparation method thereof, wherein the preparation method comprises the following steps: preparing sulfhydrylation carboxymethyl chitosan, mixing the sulfhydrylation carboxymethyl chitosan with four-arm polyvinyl alcohol norbornene, adding a photoinitiator and deionized water into the mixture, uniformly mixing to prepare a hydrogel precursor solution, carrying out ultraviolet irradiation on the hydrogel precursor solution to prepare a hydrogel matrix, and loading the hydrogel matrix with a drug to prepare the bioactive hydrogel. The hydrogel is formed by adopting a photopolymerization method, and has the advantages of rapidness and convenience; the prepared hydrogel has good biocompatibility and safe use, has certain mechanical strength, moderate viscosity when acting on a wound, can not cause secondary injury to the wound when being changed and pulled, has the functions of stopping bleeding, resisting bacteria and promoting cell proliferation and tissue regeneration, can accelerate the healing of the wound, and effectively solves the problems of the hydrogel in the prior art.

Description

Translated fromChinese

一种生物活性水凝胶及其制备方法A kind of bioactive hydrogel and preparation method thereof

技术领域technical field

本发明属于水凝胶技术领域，具体涉及一种生物活性水凝胶及其制备方法。The invention belongs to the field of hydrogel technology, and in particular relates to a bioactive hydrogel and a preparation method thereof.

背景技术Background technique

皮肤伤口愈合是皮肤损伤后组织修复和再生的一个复杂的动态过程。这个过程包括四个连续且相互重叠的阶段：止血阶段、炎症阶段、细胞增殖阶段，和组织再生阶段。相比于纱布、棉垫、合成纤维等传统伤口敷料，目前使用的半透膜、水胶体、水凝胶等新型伤口敷料允许氧气和水蒸气透过，阻碍细菌入侵，结合载入抗菌、抗炎等药物使得新型伤口敷料具备良好的生物活性更利于促进伤口愈合。其中，水凝胶具有与细胞外基质相似的微观结构，同时由于其较强亲水性，可通过吸收伤口渗出物以保持伤口周围的湿润环境，从而降低伤口感染的风险、促进伤口愈合而备受关注。Skin wound healing is a complex dynamic process of tissue repair and regeneration after skin injury. This process consists of four consecutive and overlapping phases: the hemostatic phase, the inflammatory phase, the cell proliferation phase, and the tissue regeneration phase. Compared with traditional wound dressings such as gauze, cotton pads, and synthetic fibers, new wound dressings such as semi-permeable membranes, hydrocolloids, and hydrogels currently used allow oxygen and water vapor to pass through, hinder bacterial invasion, and combine antibacterial and antibacterial properties. Drugs such as inflammation make the new wound dressing have good biological activity, which is more conducive to promoting wound healing. Among them, hydrogel has a microstructure similar to that of extracellular matrix, and because of its strong hydrophilicity, it can absorb wound exudate to maintain a moist environment around the wound, thereby reducing the risk of wound infection and promoting wound healing. much attention.

聚乙二醇(Polyethylene glycol,PEG)具有良好的水溶性、生物相容性等特性，且易于官能化。官能化PEG利用含有的活性基团与药物分子(包括蛋白药物和有机小分子药物)、或其他生物分子以及生物材料等通过共价键进行偶联，发挥药物或其他生物分子的生物活性功能。PEG基水凝胶在过去的几十年中已被广泛应用于生物医学领域，在伤口敷料中的应用亦较为普遍，例如止血敷料，抗菌敷料，载药敷料等。但在应用过程中仍然存在以下问题：Polyethylene glycol (PEG) has good water solubility, biocompatibility and other characteristics, and is easy to functionalize. Functionalized PEG utilizes the active groups contained in it to couple with drug molecules (including protein drugs and organic small molecule drugs), or other biomolecules and biomaterials through covalent bonds to exert the biologically active functions of drugs or other biomolecules. PEG-based hydrogels have been widely used in the biomedical field in the past few decades, and are also widely used in wound dressings, such as hemostatic dressings, antibacterial dressings, drug-loaded dressings, etc. However, the following problems still exist during the application process:

(1)凝胶制备过程加入的促交联物质，可能产生细胞毒性且不易去除，影响水凝胶的生物相容性。例如在制备过程中使用的交联剂、引发剂，或催化剂(如有机试剂，金属物质等)等物质，简单的物理方法难以将其去除，导致抑制了细胞的增殖与迁移，生物相容性较差。(1) The crosslinking-promoting substances added in the gel preparation process may produce cytotoxicity and are not easy to remove, affecting the biocompatibility of the hydrogel. For example, substances such as cross-linking agents, initiators, or catalysts (such as organic reagents, metal substances, etc.) used in the preparation process are difficult to remove by simple physical methods, resulting in inhibition of cell proliferation and migration, biocompatibility poor.

(2)PEG基水凝胶机械性能不佳。水凝胶应用于伤口敷料时，应具备与皮肤相当的弹性模量，符合伤口部位的机械力学状况。但纯粹的PEG基水凝胶质地脆，易断裂，粘性不强，在伤口敷料等临床应用中受到较大限制。(2) PEG-based hydrogels have poor mechanical properties. When the hydrogel is used as a wound dressing, it should have an elastic modulus comparable to that of the skin and conform to the mechanical conditions of the wound site. However, the pure PEG-based hydrogel is brittle, easy to break, and not very viscous, which is greatly limited in clinical applications such as wound dressings.

(3)凝胶功能较为单一，应用场景受限。常见伤口类型包括无菌切口、可能污染的伤口和污染伤口三种。目前，大多数水凝胶的设计缺乏普适性，一种凝胶只作用于一类伤口，无法适用于多种类型伤口的修复。(3) The function of the gel is relatively single, and the application scenarios are limited. Common wound types include sterile incisions, possibly contaminated wounds, and contaminated wounds. At present, the design of most hydrogels lacks universality. One gel only acts on one type of wound, and cannot be applied to the repair of many types of wounds.

为加速伤口愈合的整个过程以及避免愈合期间出现进一步的出血或感染等情况，研发具有止血、抗氧化、抗菌、抗炎、促愈合等多功能且生物相容性良好的伤口敷料来修复皮肤损伤部位非常重要。In order to accelerate the whole process of wound healing and avoid further bleeding or infection during the healing period, a multifunctional and biocompatible wound dressing with hemostasis, anti-oxidation, antibacterial, anti-inflammatory, and healing promotion has been developed to repair skin damage Parts are very important.

发明内容Contents of the invention

针对现有技术中存在的上述问题，本发明提供一种生物活性水凝胶及其制备方法，该水凝胶采用光聚合的方法进行成胶，具有快速方便的优点；制得的水凝胶生物相容性好，使用安全，且该水凝胶具有一定的机械强度，作用于伤口时，粘性适中，更换撕拉时不会对伤口造成二次伤害，且该水凝胶具有止血、抗菌、促进细胞和组织再生的作用，可加速伤口的愈合，有效解决现有技术中水凝胶存在的问题。Aiming at the above-mentioned problems existing in the prior art, the present invention provides a kind of bioactive hydrogel and preparation method thereof, and this hydrogel adopts the method for photopolymerization to carry out gelation, has the advantage of fast and convenient; The prepared hydrogel Good biocompatibility, safe to use, and the hydrogel has a certain mechanical strength. When acting on the wound, the viscosity is moderate. , promoting cell and tissue regeneration, can accelerate wound healing, and effectively solve the problems existing in the hydrogel in the prior art.

为实现上述目的，本发明解决其技术问题所采用的技术方案是：In order to achieve the above object, the technical solution adopted by the present invention to solve the technical problems is:

一种生物活性水凝胶的制备方法，包括以下步骤：A preparation method of bioactive hydrogel, comprising the following steps:

制备巯基化羧甲基壳聚糖，将其与四臂聚乙烯醇降冰片烯混合，向混合物中添加光引发剂和去离子水，混匀，制得水凝胶前体溶液，对水凝胶前体溶液进行紫外照射，制水凝胶基体，将水凝胶基体负载药物，制得生物活性水凝胶。Prepare mercaptolated carboxymethyl chitosan, mix it with four-arm polyvinyl alcohol norbornene, add photoinitiator and deionized water to the mixture, and mix well to obtain a hydrogel precursor solution. The gel precursor solution is irradiated with ultraviolet rays to prepare a hydrogel matrix, and the hydrogel matrix is loaded with drugs to prepare a biologically active hydrogel.

上述方案中，将巯基化羧甲基壳聚糖与四臂聚乙烯醇降冰片烯混合，两者之间通过硫醇-烯光点击化学反应得到水凝胶聚合物，该水凝胶的弹性模量与人体皮肤一致，可用于制备伤口敷料，适合于多种不同的伤口类型；硫醇-烯光点击化学反应过程使得该水凝胶的制备过程具有制备过程简单，成胶更快的优点。In the above scheme, mercaptolated carboxymethyl chitosan is mixed with four-arm polyvinyl alcohol norbornene, and a hydrogel polymer is obtained through a thiol-ene photoclick chemical reaction between the two. The elasticity of the hydrogel The modulus is consistent with that of human skin, which can be used to prepare wound dressings and is suitable for many different types of wounds; the thiol-ene photo-click chemical reaction process makes the preparation process of the hydrogel simple and the gelation is faster .

进一步地，巯基化羧甲基壳聚糖采用如下方法制备：Further, thiol carboxymethyl chitosan is prepared by the following method:

(1)将硫代乙醇酸和催化剂混合，向其中添加N,N-二甲基甲酰胺，然后搅拌反应10h以上，得反应液；(1) Mix thioglycolic acid and a catalyst, add N,N-dimethylformamide therein, and then stir and react for more than 10 hours to obtain a reaction liquid;

(2)制备羧甲基壳聚糖水溶液，然后向其中添加步骤(1)中的反应液，于黑暗环境中搅拌反应10h以上，然后将反应产物进行透析，干燥，制得巯基化羧甲基壳聚糖。(2) Prepare an aqueous solution of carboxymethyl chitosan, then add the reaction solution in step (1) to it, stir and react for more than 10h in a dark environment, then dialyze the reaction product, and dry it to obtain mercaptolated carboxymethyl Chitosan.

上述方案中，以羧甲基壳聚糖溶液和硫代乙醇酸为原料，在催化剂的作用下活化硫代乙醇酸的羧基，使其与羧甲基壳聚糖的氨基进行反应，得到接枝了多个巯基的巯基化羧甲基壳聚糖。采用透析的方式去除未参与反应的原料，制得纯度更高的产品，所制备的巯基化羧甲基壳聚糖的化学结构式如下：In the above scheme, carboxymethyl chitosan solution and thioglycolic acid are used as raw materials, and the carboxyl group of thioglycolic acid is activated under the action of a catalyst to react with the amino group of carboxymethyl chitosan to obtain grafted Thiolated carboxymethyl chitosan with multiple thiol groups. Dialysis is used to remove raw materials that do not participate in the reaction to obtain a product with higher purity. The chemical structural formula of the prepared mercaptocarboxymethyl chitosan is as follows:

由于羧甲基壳聚糖为聚合物，该聚合物中含有多个上述结构的链段，巯基化反应过程中，羧甲基壳聚糖中至少一个链段或全部链段中发生巯基化反应，因此，X限定为-H或

Since carboxymethyl chitosan is a polymer, which contains multiple chain segments of the above structure, during the thiolation reaction, at least one or all of the chain segments in carboxymethyl chitosan undergoes thiolation reaction , therefore, X is limited to -H or

进一步地，催化剂为1-乙基-(3-二甲基氨基丙基)碳酰二亚胺和N-羟基琥珀酰亚胺。Further, the catalyst is 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

进一步地，水凝胶前体溶液中四臂聚乙烯醇降冰片烯的质量百分浓度占比为4-12％，所述巯基化羧甲基壳聚糖的质量百分占比为1.5-8％，所述引发剂的占比为0.005-5％，其余为去离子水。Further, the mass percent concentration of four-arm polyvinyl alcohol norbornene in the hydrogel precursor solution is 4-12%, and the mass percent of the mercaptolated carboxymethyl chitosan is 1.5- 8%, the proportion of the initiator is 0.005-5%, and the rest is deionized water.

进一步地，巯基化羧甲基壳聚糖中巯基与四臂聚乙烯醇降冰片烯中双键的摩尔比为1-3:2-4。Further, the molar ratio of the sulfhydryl group in the mercaptolated carboxymethyl chitosan to the double bond in the four-arm polyvinyl alcohol norbornene is 1-3:2-4.

进一步地，巯基化羧甲基壳聚糖的分子量为5万-10万。Further, the molecular weight of the mercaptolated carboxymethyl chitosan is 50,000-100,000.

进一步地，光引发剂包括I2959、LAP和I1173中的至少一种。Further, the photoinitiator includes at least one of I2959, LAP and I1173.

进一步地，药物为蛋白类药物或抗生素类药物。Further, the drug is protein drug or antibiotic drug.

进一步地，药物通过浸泡溶胀的方式进行负载。Furthermore, the drug is loaded by soaking and swelling.

一种生物活性水凝胶，采用上述的方法制得。A bioactive hydrogel is prepared by the above-mentioned method.

本发明所产生的有益效果为：The beneficial effects produced by the present invention are:

1、本发明中在壳聚糖上引入巯基，制得巯基化羧甲基壳聚糖，既保留了壳聚糖自身具备的优异的生物相容性、止血性、抗菌性、生物降解性、生物粘附性以及抗氧化能力，还能作为交联剂，在与聚乙烯醇降冰片烯反应后，调节聚乙烯醇基水凝胶的机械性能，通过调整各成分的使用比例，获得与人体皮肤弹性模量相匹配的水凝胶。1. In the present invention, sulfhydryl groups are introduced into chitosan to obtain thiolated carboxymethyl chitosan, which not only retains the excellent biocompatibility, hemostasis, antibacterial properties, biodegradability, and Bioadhesion and anti-oxidation ability can also be used as a cross-linking agent to adjust the mechanical properties of polyvinyl alcohol-based hydrogel after reacting with polyvinyl alcohol norbornene. Hydrogels with skin elastic modulus matching.

2、本申请中的水凝胶是通过均聚型的双键断裂以及紫外光引发的逐步生长式硫醇-烯光点击化学反应完成。光点击化学是利用光引发剂吸收光能产生自由基，继而将聚合物链上的基团活化为自由基而发生化学反应，从而形成共价交联网络的过程。此过程发生速度快，具有时空可控性。同时，相比于传统的链式增长，逐步生长反应可以形成更为均一的网络，且因四臂聚乙二醇降冰片烯的双键加成时为均聚型，产生更少的活性氧，可有效减少氧化应激反应，避免蛋白质羰基化，减少了对于蛋白类药物的破坏。2. The hydrogel in this application is completed by the homopolymeric double bond cleavage and the stepwise growth type thiol-ene photoclick chemical reaction triggered by ultraviolet light. Photoclick chemistry is a process in which photoinitiators absorb light energy to generate free radicals, and then activate groups on the polymer chain into free radicals for chemical reactions, thereby forming a covalently crosslinked network. This process occurs quickly and is controllable in time and space. At the same time, compared with the traditional chain growth, the stepwise growth reaction can form a more uniform network, and because the double bond of the four-armed polyethylene glycol norbornene is homopolymerized, less reactive oxygen species are generated. , can effectively reduce oxidative stress response, avoid protein carbonylation, and reduce the damage to protein drugs.

3、本申请中的水凝胶基体内部具有网状结构，且具有一定的粘弹性，可以起到一定的物理阻塞作用，协同羧甲基壳聚糖的凝血作用，实现对伤口进行快速凝血止血的目的；本申请中的水凝胶采用均聚型加成的交联方式可减少活性氧的产生，羧甲基壳聚糖的阳离子具有抗氧化、抗菌的作用，PEG基水凝胶良好的防污特性也起到一定的抗菌效果，上述各种因素联合可能抑制炎症因子生成；本申请中水凝胶的三维结构与细胞外基质的结构相似，可促进组织再生；在本申请中水凝胶基体内负载具有凝血、促进细胞增殖等效果的药物，也可以辅助水凝胶基体提高止血、消炎、促进细胞再生、促进组织再生，加速伤口愈合的目的，可应用与不同类型的伤口表面。3. The hydrogel matrix in this application has a network structure inside and has a certain viscoelasticity, which can play a certain physical blocking effect, cooperate with the coagulation effect of carboxymethyl chitosan, and realize rapid coagulation and hemostasis on the wound The purpose; the hydrogel in this application adopts the cross-linking mode of homopolymerization type addition to reduce the generation of active oxygen, and the cation of carboxymethyl chitosan has anti-oxidation and antibacterial effects, and the PEG-based hydrogel has a good The anti-fouling properties also have a certain antibacterial effect. The combination of the above factors may inhibit the production of inflammatory factors; the three-dimensional structure of the hydrogel in this application is similar to the structure of the extracellular matrix, which can promote tissue regeneration; in this application, the hydrogel The gel matrix is loaded with drugs that have the effects of coagulation and cell proliferation, and can also assist the hydrogel matrix to improve hemostasis, anti-inflammation, promote cell regeneration, promote tissue regeneration, and accelerate wound healing. It can be applied to different types of wound surfaces.

附图说明Description of drawings

图1为实施例1中巯基化羧甲基壳聚糖的接枝核磁共振氢谱图；Fig. 1 is the grafted proton nuclear magnetic resonance spectrogram of mercaptolated carboxymethyl chitosan in embodiment 1;

图2为水凝胶前体溶液成胶前后的状态图；Fig. 2 is the state diagram before and after gelation of hydrogel precursor solution;

图3为水凝胶在皮肤上的粘附情况图；Figure 3 is a graph showing the adhesion of the hydrogel on the skin;

图4为水凝胶血液相容性实验结果图；Fig. 4 is the result figure of hydrogel hemocompatibility experiment;

图5为水凝胶萃取液用于细胞培养后，细胞的活性剂状态图；Figure 5 is a state diagram of the active agent of the cells after the hydrogel extract is used for cell culture;

图6为水凝胶的抗菌能力图；Figure 6 is a diagram of the antibacterial ability of the hydrogel;

图7为水凝胶用于全皮层缺损伤口时的治疗情况图；Figure 7 is a diagram of the treatment situation when the hydrogel is used for a full-thickness defect;

图8为水凝胶用于感染伤口时的治疗情况图；Figure 8 is a diagram of the treatment situation when the hydrogel is used for infected wounds;

图9为水凝胶用于烫伤伤口时的治疗情况图。Fig. 9 is a diagram of the treatment situation when the hydrogel is used for a scald wound.

具体实施方式Detailed ways

为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明，即所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, and are not intended to limit the present invention, that is, the described embodiments are only some of the embodiments of the present invention, but not all of the embodiments.

因此，以下对提供的本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施例。基于本发明的实施例，本领域技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。Accordingly, the following detailed description of the embodiments of the invention provided is not intended to limit the scope of the claimed invention, but represents only selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative efforts belong to the protection scope of the present invention.

需要说明的是，术语“第一”和“第二”等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括要素的过程、方法、物品或者设备中还存在另外的相同要素。It should be noted that relative terms such as the terms "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any relationship between these entities or operations. There is no such actual relationship or order between them. Furthermore, the term "comprises", "comprises" or any other variation thereof is intended to cover a non-exclusive inclusion such that a process, method, article, or apparatus comprising a set of elements includes not only those elements, but also includes elements not expressly listed. other elements of or also include elements inherent in such a process, method, article, or device. Without further limitations, an element defined by the phrase "comprising a ..." does not preclude the presence of additional identical elements in the process, method, article, or apparatus that includes the element.

下面结合实施例对本发明的特征和性能作进一步的详细描述。The characteristics and performance of the present invention will be further described in detail below in conjunction with the examples.

实施例1Example 1

一种生物活性水凝胶，其制备方法包括以下步骤：A bioactive hydrogel, the preparation method of which comprises the following steps:

(1)合成巯基化羧甲基壳聚糖(1) Synthesis of thiolated carboxymethyl chitosan

将1mL 10mol/L硫代乙醇酸分析纯化学品加入到0.5g 1-乙基-(3-二甲基氨基丙基)碳酰二亚胺和0.3g N-羟基琥珀酰亚胺的混合物中，补入足量的N,N-二甲基甲酰胺，于磁力搅拌器上反应12h，活化羧基；称取1g羧甲基壳聚糖(MW＝50000g/mol)于锥形瓶中，加入40mL超纯水，超声或搅拌完全溶解后，加入活化的硫代乙醇酸，于黑暗中置于磁力搅拌器上反应12h，得到巯基化羧甲基壳聚糖产物以及未完全发生反应的原料物质；将得到的产物置于合适大小的透析袋中进行产物纯化，去除未反应的原材料，透析完成后，将纯化产物置于-20℃条件下进行预冷12h，最后在真空干燥剂中冷冻干燥，得到纯化的巯基化羧甲基壳聚糖；Add 1mL of 10mol/L thioglycolic acid analytically pure chemical to a mixture of 0.5g 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.3g N-hydroxysuccinimide , add a sufficient amount of N,N-dimethylformamide, react on a magnetic stirrer for 12h, and activate the carboxyl group; weigh 1g carboxymethyl chitosan (MW=50000g/mol) in a conical flask, add 40mL of ultrapure water, ultrasonic or stirring completely dissolved, added activated thioglycolic acid, placed in the dark on a magnetic stirrer for 12h reaction, to obtain mercapto-carboxymethyl chitosan products and incompletely reacted raw materials ; Place the obtained product in a dialysis bag of a suitable size for product purification to remove unreacted raw materials. After the dialysis is completed, place the purified product at -20°C for pre-cooling for 12 hours, and finally freeze-dry it in a vacuum desiccant , to obtain purified thiolated carboxymethyl chitosan;

(2)四臂聚乙二醇降冰片烯/巯基化羧甲基壳聚糖水凝胶前体溶液的制备(2) Preparation of four-arm polyethylene glycol norbornene/mercaptocarboxymethyl chitosan hydrogel precursor solution

将5mg四臂聚乙二醇降冰片烯、3.75mg巯基化羧甲基壳聚糖、6.75μg光引发剂I2959以及去离子水混合为100μL水凝胶的前体溶液，其中双键和巯基的摩尔比为1:2；Mix 5 mg four-arm polyethylene glycol norbornene, 3.75 mg thiolated carboxymethyl chitosan, 6.75 μg photoinitiator I2959, and deionized water to form a 100 μL hydrogel precursor solution, in which the double bond and thiol The molar ratio is 1:2;

(3)伤口部位原位成胶(3) In-situ gelation at the wound site

在全皮层损伤伤口处加入40μL水凝胶前体溶液，365nm紫外光照5分钟完成凝胶过程；载入10μL 20μg/mL表皮生长因子(EGF)和10μL 5μg/mL碱性成纤维细胞生长因子(bFGF)，待水凝胶状态稳定后，使用透明敷贴保证水凝胶不轻易脱落。Add 40 μL hydrogel precursor solution to the full-thickness injury wound, and 365 nm ultraviolet light for 5 minutes to complete the gel process; load 10 μL 20 μg/mL epidermal growth factor (EGF) and 10μL 5 μg/mL basic fibroblast growth factor ( bFGF), after the hydrogel is in a stable state, use a transparent patch to ensure that the hydrogel does not fall off easily.

实施例2Example 2

将5mg四臂聚乙二醇降冰片烯、1.875mg巯基化羧甲基壳聚糖、6.75μg光引发剂I2959以及去离子水混合为100μL水凝胶的前体溶液，其中四臂聚乙二醇降冰片烯中双键和巯基化羧甲基壳聚糖中巯基的摩尔比为1:4；Mix 5 mg of four-armed polyethylene glycol norbornene, 1.875 mg of thiolated carboxymethyl chitosan, 6.75 μg of photoinitiator I2959, and deionized water to form a 100 μL hydrogel precursor solution, in which four-armed polyethylene glycol The molar ratio of the double bond in the alcohol norbornene and the sulfhydryl group in the thiolated carboxymethyl chitosan is 1:4;

(3)伤口部位原位成胶(3) In-situ gelation at the wound site

实施例3Example 3

将5mg四臂聚乙二醇降冰片烯、5.625mg巯基化羧甲基壳聚糖、6.75μg光引发剂I2959以及去离子水混合为100μL水凝胶的前体溶液，其中双键和巯基的摩尔比为3:4；Mix 5 mg four-arm polyethylene glycol norbornene, 5.625 mg thiolated carboxymethyl chitosan, 6.75 μg photoinitiator I2959, and deionized water to form a 100 μL hydrogel precursor solution, in which the double bonds and thiols The molar ratio is 3:4;

(3)伤口部位原位成胶(3) In-situ gelation at the wound site

实施例4Example 4

将1mL 10mol/L硫代乙醇酸分析纯化学品加入到0.5g 1-乙基-(3-二甲基氨基丙基)碳酰二亚胺和0.3g N-羟基琥珀酰亚胺的混合物中，补入足量的N,N-二甲基甲酰胺，于磁力搅拌器上反应12h，活化羧基；称取1g羧甲基壳聚糖(MW＝100000g/mol)于锥形瓶中，加入40mL超纯水，超声或搅拌完全溶解后，加入活化的硫代乙醇酸，于黑暗中置于磁力搅拌器上反应12h，得到巯基化羧甲基壳聚糖产物以及未完全发生反应的原料物质；将得到的产物置于合适大小的透析袋中进行产物纯化，去除未反应的原材料，透析完成后，将纯化产物置于-20℃条件下进行预冷12h，最后在真空干燥剂中冷冻干燥，得到纯化的巯基化羧甲基壳聚糖；Add 1mL of 10mol/L thioglycolic acid analytically pure chemical to a mixture of 0.5g 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.3g N-hydroxysuccinimide , add a sufficient amount of N,N-dimethylformamide, react on a magnetic stirrer for 12h, and activate the carboxyl group; weigh 1g carboxymethyl chitosan (MW=100000g/mol) in a conical flask, add 40mL of ultrapure water, ultrasonic or stirring completely dissolved, added activated thioglycolic acid, placed in the dark on a magnetic stirrer for 12h reaction, to obtain mercapto-carboxymethyl chitosan products and incompletely reacted raw materials ; Place the obtained product in a dialysis bag of a suitable size for product purification to remove unreacted raw materials. After the dialysis is completed, place the purified product at -20°C for pre-cooling for 12 hours, and finally freeze-dry it in a vacuum desiccant , to obtain purified thiolated carboxymethyl chitosan;

将5mg四臂聚乙二醇降冰片烯、1.875mg巯基化羧甲基壳聚糖、6.75μg光引发剂I2959以及去离子水混合为100μL水凝胶的前体溶液，其中双键和巯基的摩尔比为1:4；Mix 5 mg of four-arm polyethylene glycol norbornene, 1.875 mg of thiolated carboxymethyl chitosan, 6.75 μg of photoinitiator I2959, and deionized water to form a 100 μL hydrogel precursor solution, in which the double bond and thiol The molar ratio is 1:4;

(3)伤口部位原位成胶(3) In-situ gelation at the wound site

实施例5Example 5

(3)伤口部位原位成胶(3) In-situ gelation at the wound site

实施例6Example 6

(3)伤口部位原位成胶(3) In-situ gelation at the wound site

实施例7Example 7

将1mL 10mol/L硫代乙醇酸分析纯化学品加入到0.5g 1-乙基-(3-二甲基氨基丙基)碳酰二亚胺和0.3g N-羟基琥珀酰亚胺的混合物中，补入足量的N,N-二甲基甲酰胺，于磁力搅拌器上反应12h，活化羧基；称取1g羧甲基壳聚糖(MW＝50000g/mol)于锥形瓶中，加入40mL超纯水，超声或搅拌完全溶解后，加入活化的硫代乙醇酸，于黑暗中置于磁力搅拌器上反应12h，得到巯基化羧甲基壳聚糖产物以及未完全发生反应的原料物质；将得到的产物置于合适大小的透析袋中进行产物纯化，去除未反应的原材料。透析完成后，将纯化产物置于-20℃条件下进行预冷12h，最后在真空干燥剂中冷冻干燥，得到纯化的巯基化羧甲基壳聚糖；Add 1mL of 10mol/L thioglycolic acid analytically pure chemical to a mixture of 0.5g 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.3g N-hydroxysuccinimide , add a sufficient amount of N,N-dimethylformamide, react on a magnetic stirrer for 12h, and activate the carboxyl group; weigh 1g carboxymethyl chitosan (MW=50000g/mol) in a conical flask, add 40mL of ultrapure water, ultrasonic or stirring completely dissolved, added activated thioglycolic acid, placed in the dark on a magnetic stirrer for 12h reaction, to obtain mercapto-carboxymethyl chitosan products and incompletely reacted raw materials ; Place the obtained product in a dialysis bag of appropriate size for product purification to remove unreacted raw materials. After the dialysis was completed, the purified product was pre-cooled at -20°C for 12 hours, and finally freeze-dried in a vacuum desiccant to obtain purified thiolated carboxymethyl chitosan;

(3)感染伤口部位原位成胶(3) In situ gelation at the infected wound site

在感染伤口处加入40μL水凝胶前体溶液，365nm紫外光照5分钟完成凝胶过程；载入10μL 20μg/mL盐酸多西环素，待水凝胶状态稳定后，使用透明敷贴保证水凝胶不轻易脱落。Add 40 μL of hydrogel precursor solution to the infected wound, and irradiate with 365 nm ultraviolet light for 5 minutes to complete the gelation process; load 10 μL of 20 μg/mL doxycycline hydrochloride, and after the hydrogel is stable, use a transparent applicator to ensure the hydrogel The glue does not fall off easily.

实施例8Example 8

(2)四臂聚乙二醇降冰片烯/巯基化羧甲基壳聚糖水凝胶的制备(2) Preparation of four-arm polyethylene glycol norbornene/mercaptocarboxymethyl chitosan hydrogel

将5mg四臂聚乙二醇降冰片烯、3.75mg巯基化羧甲基壳聚糖、6.75μg光引发剂I2959以及去离子水混合为100μL水凝胶的前体溶液，其中双键和巯基的摩尔比为1:2；在紫外光照射下成胶，成胶完成后载入10μL 20μg/mL表皮生长因子(EGF)和10μL 5μg/mL碱性成纤维细胞生长因子(bFGF)；Mix 5 mg four-arm polyethylene glycol norbornene, 3.75 mg thiolated carboxymethyl chitosan, 6.75 μg photoinitiator I2959, and deionized water to form a 100 μL hydrogel precursor solution, in which the double bond and thiol The molar ratio is 1:2; the gel is formed under ultraviolet light irradiation, and 10 μL of 20 μg/mL epidermal growth factor (EGF) and 10 μL of 5 μg/mL basic fibroblast growth factor (bFGF) are loaded after the gelation is completed;

(3)烫伤伤口部位的应用(3) Application of scald wound site

在烫伤伤口处放置水凝胶，然后使用透明敷贴保证水凝胶不轻易脱落。Put the hydrogel on the burn wound, and then use a transparent plaster to ensure that the hydrogel will not fall off easily.

实施例9Example 9

将5mg四臂聚乙二醇降冰片烯、3.75mg巯基化羧甲基壳聚糖、100μg光引发剂I2959以及去离子水混合为100μL水凝胶的前体溶液，其中双键和巯基的摩尔比为1:2；在紫外光照射下成胶，成胶完成后载入10μL 12nM组织因子(TF)；Mix 5 mg of four-armed polyethylene glycol norbornene, 3.75 mg of thiolated carboxymethyl chitosan, 100 μg of photoinitiator I2959, and deionized water to form a 100 μL hydrogel precursor solution, in which moles of double bonds and thiols The ratio is 1:2; the gel is formed under ultraviolet light, and 10 μL of 12nM tissue factor (TF) is loaded after the gel is completed;

(3)出血伤口部位的应用(3) Application of bleeding wound site

在出血伤口处放置水凝胶，利用水凝胶的物理阻隔作用和TF的外源性快速凝血途径进行止血。Place the hydrogel on the bleeding wound, and use the physical barrier effect of the hydrogel and the exogenous rapid coagulation pathway of TF for hemostasis.

试验例Test case

1、核磁共振波谱分析1. NMR spectrum analysis

对本发明实施例1制备得到的巯基化羧甲基壳聚糖进行核磁共振波谱分析。MestReNova软件的分析结果如图1所示，巯基特征峰出现在2.9ppm的位置，表明巯基的成功接枝。The mercaptolated carboxymethyl chitosan prepared in Example 1 of the present invention was subjected to nuclear magnetic resonance spectrum analysis. The analysis result of MestReNova software is shown in Figure 1, the characteristic peak of thiol appears at the position of 2.9ppm, indicating the successful grafting of thiol.

2、四臂聚乙二醇降冰片烯/巯基化羧甲基壳聚糖水凝胶状态变化2. State change of four-armed polyethylene glycol norbornene/mercaptocarboxymethyl chitosan hydrogel

四臂聚乙二醇降冰片烯/巯基化羧甲基壳聚糖水凝胶制备过程中，在紫外光照前后分别记录水凝胶物理状态的变化，如图2所示，可以明确看出，前体溶液为液体状态转变为固体的水凝胶。During the preparation process of the four-armed polyethylene glycol norbornene/mercapto carboxymethyl chitosan hydrogel, the changes in the physical state of the hydrogel were recorded before and after ultraviolet light, as shown in Figure 2. It can be clearly seen that the former The body solution is a hydrogel that changes from a liquid state to a solid.

3、皮肤粘附能力3. Skin adhesion ability

直接将制好的水凝胶贴在人体皮肤上，如图3所示，在倒置时水凝胶也具有一定的粘性，能够与皮肤有较好的贴合度。The prepared hydrogel is directly pasted on the human skin, as shown in Figure 3, the hydrogel also has a certain viscosity when inverted, and can have a good fit with the skin.

4、体外生物相容性检测4. In vitro biocompatibility testing

将水凝胶与兔全血共孵育后，离心，结果见图4，结果表明水凝胶不会导致大量的红细胞破裂，即血液相容性良好。此外，通过细胞实验验证四臂聚乙二醇降冰片烯/巯基化羧甲基壳聚糖水凝胶良好的细胞相容性。After the hydrogel was co-incubated with rabbit whole blood, it was centrifuged, and the results are shown in Figure 4. The results show that the hydrogel will not cause a large number of red blood cells to rupture, that is, the blood compatibility is good. In addition, the good cytocompatibility of the four-armed polyethylene glycol norbornene/mercaptocarboxymethyl chitosan hydrogel was verified by cell experiments.

将小鼠皮肤成纤维细胞株L929接种于空白孔板，使用水凝胶萃取液培养3天，经罗丹明细胞质染色后进行显微观察，结果见图5，实验结果得出细胞折光性强，胞质饱满，增殖能力较强，细胞状态未发生明显变化，且保持高活性，细胞相容性良好。The mouse skin fibroblast cell line L929 was inoculated in a blank well plate, cultured with hydrogel extract for 3 days, and subjected to microscopic observation after rhodamine cytoplasmic staining. The results are shown in Figure 5. The experimental results show that the cells have strong refraction, The cytoplasm is full, the proliferative ability is strong, the cell state does not change significantly, and maintains high activity, and the cell compatibility is good.

5、抗菌能力5. Antibacterial ability

通过对于大肠杆菌和金黄色葡萄球菌的抗性实验验证四臂聚乙二醇降冰片烯/巯基化羧甲基壳聚糖水凝胶良好的抗菌能力。于孔板中成胶后，将10⁵CFU/mL的菌液滴加在水凝胶上，细菌培养箱中孵育4小时，冲洗后的上清液涂布于细菌培养基上，过夜孵育后进行观察及计数。如图6所示，实验结果表明相比于对照组，水凝胶组做到了完全抗菌。The good antibacterial ability of the four-armed polyethylene glycol norbornene/mercaptolated carboxymethyl chitosan hydrogel was verified by the resistance experiments against Escherichia coli and Staphylococcus aureus. After gelling in the orifice plate, add 10⁵ CFU/mL of bacterial solution dropwise on the hydrogel, incubate in the bacterial incubator for 4 hours, apply the washed supernatant on the bacterial medium, and after overnight incubation Observe and count. As shown in Figure 6, the experimental results show that compared with the control group, the hydrogel group achieved complete antibacterial.

6、体内生物相容性检测6. In vivo biocompatibility testing

通过三种动物伤口模型评价四臂聚乙二醇降冰片烯/巯基化羧甲基壳聚糖水凝胶的体内生物相容性及促愈合能力。对照组为PBS溶液，商用组为液体敷料，实验组为四臂聚乙二醇降冰片烯/巯基化羧甲基壳聚糖水凝胶，以伤口尺寸、伤口感染(化脓)程度以及伤口愈合时间为指标进行评价。如图7所示，全皮层缺损伤口的愈合图表明，水凝胶组的开始愈合的时间较早，促愈合能力在PBS组之上，甚至略优于商用组，表明四臂聚乙二醇降冰片烯/巯基化羧甲基壳聚糖水凝胶对于普通创面有着优秀的促愈合作用；如图8所示，在伤口部位加入了金黄色葡萄球菌模拟感染伤口时，商用组和PBS组的伤口部位均出现了明显的脓液，而水凝胶组一直保持伤口干净，稳步愈合，表明四臂聚乙二醇降冰片烯/巯基化羧甲基壳聚糖水凝胶对于感染创面有着优秀的抗菌、抗炎、以及促愈合作用；如图9所示，烫伤伤口模型可以看出水凝胶组的伤口与其他两组相比，从初期便更快地愈合，最终愈合部位的疤痕区域面积小且深度较浅。因此，本发明中的四臂聚乙二醇降冰片烯/巯基化羧甲基壳聚糖水凝胶作为一种高含水量的伤口敷料，也同样适用于烫伤伤口。The in vivo biocompatibility and healing-promoting ability of four-armed polyethylene glycol norbornene/mercaptocarboxymethyl chitosan hydrogel were evaluated by three animal wound models. The control group was PBS solution, the commercial group was liquid dressing, and the experimental group was four-arm polyethylene glycol norbornene/mercaptocarboxymethyl chitosan hydrogel. The wound size, wound infection (purulence) degree and wound healing time Evaluate the indicators. As shown in Figure 7, the healing chart of the full-thickness defect shows that the healing time of the hydrogel group is earlier, and the ability to promote healing is higher than that of the PBS group, and even slightly better than the commercial group, indicating that the four-armed polyethylene glycol group Norbornene/mercapto carboxymethyl chitosan hydrogel has an excellent healing-promoting effect on common wounds; as shown in Figure 8, when Staphylococcus aureus was added to the wound site to simulate wound infection, the commercial group and PBS group Obvious pus appeared at the wound site, while the hydrogel group kept the wound clean and healed steadily, indicating that the four-armed polyethylene glycol norbornene/mercaptocarboxymethyl chitosan hydrogel has an excellent effect on infected wounds. Antibacterial, anti-inflammatory, and healing-promoting effects; as shown in Figure 9, the scald wound model shows that the wound in the hydrogel group heals faster from the initial stage compared with the other two groups, and the scar area at the final healing site is small And the depth is shallow. Therefore, the four-arm polyethylene glycol norbornene/mercaptolated carboxymethyl chitosan hydrogel of the present invention is also suitable for scald wounds as a wound dressing with high water content.

Claims

1. A method for preparing a bioactive hydrogel, comprising the steps of:

preparing sulfhydrylation carboxymethyl chitosan, mixing the sulfhydrylation carboxymethyl chitosan with four-arm polyvinyl alcohol norbornene, adding a photoinitiator and deionized water into the mixture, uniformly mixing to prepare a hydrogel precursor solution, carrying out ultraviolet irradiation on the hydrogel precursor solution to prepare a hydrogel matrix, and loading the hydrogel matrix with a drug to prepare the bioactive hydrogel.

2. The method for preparing the bioactive hydrogel according to claim 1, wherein the sulfhydryl carboxymethyl chitosan is prepared by the following method:

(1) Thioglycollic acid and a catalyst are mixed, N-dimethylformamide is added into the mixture, and then the mixture is stirred and reacted for more than 10 hours to obtain a reaction solution;

(2) Preparing carboxymethyl chitosan water solution, adding the reaction solution in the step (1), stirring and reacting for more than 10 hours in a dark environment, dialyzing the reaction product, and drying to obtain the sulfhydryl carboxymethyl chitosan.

3. The method of preparing a bioactive hydrogel according to claim 2, wherein said catalyst is 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide.

4. The method for preparing a bioactive hydrogel according to claim 1, wherein the mass percentage concentration of the four-arm polyvinyl alcohol norbornene in the hydrogel precursor solution is 4-12%, the mass percentage of the sulfhydryl carboxymethyl chitosan is 1.5-8%, the mass percentage of the initiator is 0.005-5%, and the balance is deionized water.

5. The method for preparing a bioactive hydrogel according to claim 1, wherein the molar ratio of mercapto groups in the thiolated carboxymethyl chitosan to double bonds in the four-arm polyvinyl alcohol norbornene is 1-3:2-4.

6. The method for preparing a bioactive hydrogel according to claim 1, wherein the molecular weight of the thiolated carboxymethyl chitosan is 5-10 tens of thousands.

7. The method of preparing a bioactive hydrogel according to claim 1, wherein said photoinitiator comprises at least one of I2959, LAP, and I1173.

8. The method of claim 1, wherein the drug is a protein drug or an antibiotic drug.

9. The method for preparing a bioactive hydrogel according to claim 1, wherein said drug is loaded by means of soaking swelling.

10. A bioactive hydrogel, characterized in that it is produced by the method according to any one of claims 1-9.