CN113069412A - 一种用于皮肤创伤修复的可注射复合壳聚糖水凝胶的制备方法 - Google Patents
一种用于皮肤创伤修复的可注射复合壳聚糖水凝胶的制备方法 Download PDFInfo
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Abstract
本发明涉及一种用于皮肤创伤修复的可注射复合壳聚糖水凝胶的制备方法，该水凝胶主要由改性壳聚糖、内载细胞以及生物活性因子组成。首先通过乳液聚合制备得到水凝胶前体溶液，主要成分为聚N‑异丙基丙烯酰胺接枝改性的壳聚糖大分子，该水凝胶能在室温环境下呈溶液状态，在人体生理温度环境下实现快速凝胶化。通过与细胞悬液和生物活性因子的混合，所得复合溶液可以在皮肤创伤缺损部位实现原位快速凝胶化，实现细胞原位固定，不仅可以为损伤部位提供良好的支撑，还可以促进其内部细胞的粘附、增殖、分化。由于制备简单、使用方便、响应迅速、生物相容性良好，该水凝胶可用于组织修复、药物控释、细胞培养、癌症治疗等诸多生物医学相关领域。
Description
技术领域
本发明涉及一种壳聚糖基可注射水凝胶，尤其涉及一种用于皮肤创伤修复的可注射复合壳聚糖水凝胶的制备方法。
背景技术
皮肤是包在身体表面，直接同外界环境接触，具有保护、排泄、调节体温和感受外界刺激等作用的一种器官，是人体中最大的器官。皮肤由多种细胞和组织构成，具有多层复杂结构，其每层的组成和功能都不尽相同。由外伤或烧伤等原因引起的皮肤损伤修复，特别是深度的全皮肤创伤，会导致皮肤的多层结构被破坏，多种细胞及蛋白大量流失。在避免细菌、真菌等病原体黏附扩增使组织感染的前体下，如何有效地使细胞在皮肤损伤部位富集、增殖、分化，促进皮肤损伤修复，恢复皮肤原来的形态及功能，一直是临床医学的一大难题。
理想的皮肤创伤修复再生材料不仅需要合适的力学强度在皮肤损伤部位提供短期支撑，还应该促进细胞黏附、增殖、分化、矿化。水凝胶材料具有高含水量、与细胞外基质相似的结构、可调控的力学性能，使其在创伤修复方面有着良好的应用前景。与传统水凝胶相比，智能水凝胶因其能对外界环境变化产生响应而受到更多关注。温度敏感水凝胶是一种对外界温度变化产生响应的水凝胶，具有可注射性，温敏水凝胶中亲水基团与疏水基团的存在，可以使温敏凝胶在温度改变时发生相转变，由液态转变为固态。具有良好生物相容性的温敏可注射水凝胶可以在组织修复领域发挥极大地作用。
壳聚糖是一种天然氨基多糖，来源于生物体，是虾壳、螃蟹壳的重要组成部分。壳聚糖不仅来源广泛，还具有优异的生物相容性、抑菌性以及良好的生物可降解性。温敏壳聚糖水凝胶兼具水凝胶的特点和优良的本征属性，为皮肤创伤修复提供了新思路和新方法。
发明内容
本发明的目的在于针对现有技术的不足，提供一种用于皮肤创伤修复的可注射复合壳聚糖水凝胶的制备方法。本发明的用于皮肤创伤修复的可注射复合壳聚糖水凝胶，主要由改性壳聚糖、内载细胞以及生物活性因子组成。
所述改性壳聚糖为聚N-异丙基丙烯酰胺接枝改性的壳聚糖。
所述内载细胞为表皮干细胞、胚胎干细胞、间充质干细胞、脂肪干细胞、表皮细胞、内皮细胞、角质细胞中的一种或几种。
所述生物活性因子为抗菌活性因子、促细胞生长因子中的一种或几种，其中所述抗菌活性因子为醋酸氯己定CHX、银纳米颗粒、抗菌肽中的一种或几种，其中促细胞生长因子为VEGF、EGF、聚多巴胺、胶原蛋白、RGD、丝胶蛋白、丝素蛋白以及其他氨基酸或多肽中的一种或几种。
本发明所述的一种用于皮肤创伤修复的可注射复合壳聚糖水凝胶的制备方法是：
1)聚N-异丙基丙烯酰胺接枝改性的壳聚糖生物大分子的制备：按照质量计，将1-20份壳聚糖、0.1-4份酸、0.01-10份引发剂、1-20份N-异丙基丙烯酰胺、50-98份溶剂加入烧杯中，搅拌溶解均匀后，加热至40-80℃反应1-12小时，随后将溶液转移入透析袋中，用去离子水反复透析，冷冻干燥得到聚N-异丙基丙烯酰胺接枝改性的壳聚糖粉末；
2)聚N-异丙基丙烯酰胺接枝改性的壳聚糖水溶液的制备：将1-20份1)中的聚N-异丙基丙烯酰胺接枝改性的壳聚糖粉末加入80-99份PBS溶液中，低温环境下搅拌溶解均匀后得到水凝胶前体溶液。
3)可注射复合壳聚糖水凝胶溶液的制备：将权利要求1所述步骤2)中的水凝胶前体溶液进行灭菌，将10-50份灭菌后的水凝胶前体溶液、5份内载细胞的细胞悬液与0.1-5份生物活性因子低温环境下进行共混，混合均匀得到可注射复合壳聚糖水凝胶溶液；
上述技术方案中，进一步的，
所述低温环境为4℃-25℃。
所述细胞悬液中内载细胞的浓度为2万-200万/ml，细胞培养基为完全培养基或定向诱导分化培养基。
所述壳聚糖的分子量为3万-200万，脱乙酰度为65％-99％。
所述酸为盐酸、醋酸、磷酸、柠檬酸、酒石酸、丙烯酸中的一种或几种。
所述引发剂为偶氮二异丁腈、偶氮二异庚腈、偶氮二异丁酸二甲酯、过氧化氢、过硫酸铵、过硫酸钾、叔丁基过氧化氢中的一种或几种。
所述溶剂为去离子水、乙醇、DMSO中的一种或几种。
所述的可注射复合壳聚糖水凝胶溶液可用于皮肤创伤修复。
本发明的有益效果在于：
本发明首先通过乳液聚合制备得到水凝胶前体溶液，主要成分为聚N-异丙基丙烯酰胺接枝改性的壳聚糖大分子，该水凝胶具有温度敏感特性，能在室温环境下呈溶液状态，在人体生理温度环境下实现快速凝胶化。该温敏水凝胶具有优异的生物相容性，通过与细胞悬液和生物活性因子的混合，该水凝胶溶液可以在皮肤创伤部位实现原位快速凝胶化，不仅可以为损伤部位提供良好的支撑，还可以促进其内部细胞的粘附、增殖、分化。由于制备简单、使用方便、响应迅速、生物相容性良好等诸多特点，该水凝胶可用于组织修复、药物控释、细胞培养、癌症治疗等诸多生物医学相关领域，特别是应用于皮肤创伤修复再生。
具体实施方式
以下结合具体实例进一步说明本发明。
实施例1：
1)称取1.5g粘均分子量为65W、脱乙酰度为90％的壳聚糖粉末、1.75gN-异丙基丙烯酰胺、0.075g过硫酸铵，分散在95mL去离子水中，然后，加入0.2mL的盐酸，搅拌溶解均匀后，加热至60℃反应8小时，随后将溶液转移入透析袋中，用去离子水反复透析，冷冻干燥得到聚N-异丙基丙烯酰胺接枝改性的壳聚糖粉末；
2)将1-20份步骤1)中的聚N-异丙基丙烯酰胺接枝改性的壳聚糖粉末加入80-99份PBS溶液中，4℃环境下搅拌溶解均匀后得到水凝胶前体溶液；
3)将步骤2)中的水凝胶前体溶液进行灭菌，将30份灭菌后的水凝胶前体溶液、5份内载脂肪干细胞的细胞悬液与0.1份胶原蛋白于4℃环境下进行共混，混合均匀得到可注射复合壳聚糖水凝胶溶液；
实施例2：
1)称取1.5g粘均分子量为65W、脱乙酰度为90％的壳聚糖粉末、1.75gN-异丙基丙烯酰胺、0.075g过硫酸铵，分散在95mL去离子水中，然后，加入0.2mL的盐酸，搅拌溶解均匀后，加热至60℃反应8小时，随后将溶液转移入透析袋中，用去离子水反复透析，冷冻干燥得到聚N-异丙基丙烯酰胺接枝改性的壳聚糖粉末；
2)将1-20份步骤1)中的聚N-异丙基丙烯酰胺接枝改性的壳聚糖粉末加入80-99份PBS溶液中，4℃环境下搅拌溶解均匀后得到水凝胶前体溶液；
3)将步骤2)中的水凝胶前体溶液进行灭菌，将20份灭菌后的水凝胶前体溶液、5份内载表皮干细胞的细胞悬液与0.2份RGD分子于4℃环境下进行共混，混合均匀得到可注射复合壳聚糖水凝胶溶液；
实施例3：
1)称取1.5g粘均分子量为65W、脱乙酰度为90％的壳聚糖粉末、1.75gN-异丙基丙烯酰胺、0.075g过硫酸铵，分散在95mL去离子水中，然后，加入0.2mL的盐酸，搅拌溶解均匀后，加热至60℃反应8小时，随后将溶液转移入透析袋中，用去离子水反复透析，冷冻干燥得到聚N-异丙基丙烯酰胺接枝改性的壳聚糖粉末；
2)将1-20份步骤1)中的聚N-异丙基丙烯酰胺接枝改性的壳聚糖粉末加入80-99份PBS溶液中，4℃环境下搅拌溶解均匀后得到水凝胶前体溶液；
3)将步骤2)中的水凝胶前体溶液进行灭菌，将50份灭菌后的水凝胶前体溶液、5份内载胚胎干细胞的细胞悬液与0.5份CHX分子及0.5份丝素蛋白于4℃环境下进行共混，混合均匀得到可注射复合壳聚糖水凝胶溶液。
Claims (10)
1.一种用于皮肤创伤修复的可注射复合壳聚糖水凝胶，其特征在于，该水凝胶主要由改性壳聚糖、内载细胞以及生物活性因子组成，所述改性壳聚糖为聚N-异丙基丙烯酰胺接枝改性的壳聚糖；其制备方法具体包括如下步骤：
1)聚N-异丙基丙烯酰胺接枝改性的壳聚糖生物大分子的制备：按照质量计，将1-20份壳聚糖、0.1-4份酸、0.01-10份引发剂、1-20份N-异丙基丙烯酰胺、50-98份溶剂加入烧杯中，搅拌溶解均匀后，加热至40-80℃反应1-12小时，随后将溶液转移入透析袋中，用去离子水反复透析，冷冻干燥得到聚N-异丙基丙烯酰胺接枝改性的壳聚糖粉末；
2)聚N-异丙基丙烯酰胺接枝改性的壳聚糖水溶液的制备：将1-20份1)中的聚N-异丙基丙烯酰胺接枝改性的壳聚糖粉末加入80-99份PBS溶液中，低温环境下搅拌溶解均匀后得到水凝胶前体溶液；
3)可注射复合壳聚糖水凝胶溶液的制备：将步骤2)中的水凝胶前体溶液进行灭菌，将10-50份灭菌后的水凝胶前体溶液、5份内载细胞的细胞悬液与0.1-5份生物活性因子低温环境下进行共混，混合均匀得到可注射复合壳聚糖水凝胶溶液。
2.根据权利要求1所述的用于皮肤创伤修复的可注射复合壳聚糖水凝胶，其特征在于，步骤3)中所述内载细胞为表皮干细胞、胚胎干细胞、间充质干细胞、脂肪干细胞、表皮细胞、内皮细胞、角质细胞中的一种或几种。
3.根据权利要求1所述的用于皮肤创伤修复的可注射复合壳聚糖水凝胶，其特征在于，步骤3)中所述生物活性因子为抗菌活性因子、促细胞生长因子中的一种或几种，其中所述抗菌活性因子为醋酸氯己定CHX、银纳米颗粒、抗菌肽中的一种或几种，其中促细胞生长因子为VEGF、EGF、聚多巴胺、胶原蛋白、RGD、丝胶蛋白、丝素蛋白中的一种或几种。
4.根据权利要求1所述的用于皮肤创伤修复的可注射复合壳聚糖水凝胶，其特征在于，步骤2)和步骤3)中所述低温环境为4℃-25℃。
5.根据权利要求1所述的用于皮肤创伤修复的可注射复合壳聚糖水凝胶，其特征在于，步骤3)中所述细胞悬液中内载细胞的浓度为2万-200万/ml，细胞培养基为完全培养基或定向诱导分化培养基。
6.根据权利要求1所述的用于皮肤创伤修复的可注射复合壳聚糖水凝胶，其特征在于，步骤1)中所述壳聚糖的分子量为3万-200万，脱乙酰度为65％-99％。
7.根据权利要求1所述的用于皮肤创伤修复的可注射复合壳聚糖水凝胶，其特征在于，步骤1)中所述酸为盐酸、醋酸、磷酸、柠檬酸、酒石酸、丙烯酸中的一种或几种。
8.根据权利要求1所述的用于皮肤创伤修复的可注射复合壳聚糖水凝胶，其特征在于，步骤1)中所述引发剂为偶氮二异丁腈、偶氮二异庚腈、偶氮二异丁酸二甲酯、过氧化氢、过硫酸铵、过硫酸钾、叔丁基过氧化氢中的一种或几种。
9.根据权利要求1所述的用于皮肤创伤修复的可注射复合壳聚糖水凝胶，其特征在于，步骤1)中所述溶剂为去离子水、乙醇、DMSO中的一种或几种。
10.根据权利要求1所述的用于皮肤创伤修复的可注射复合壳聚糖水凝胶，其特征在于，所述的可注射复合壳聚糖水凝胶溶液用于皮肤创伤修复，将所述水凝胶溶液置于待修复皮肤创伤部位凝胶化即可。
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