CN116554505A - 一种玛咖多糖水凝胶的制备方法及其应用 - Google Patents
一种玛咖多糖水凝胶的制备方法及其应用 Download PDFInfo
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- CN116554505A CN116554505A CN202310688050.2A CN202310688050A CN116554505A CN 116554505 A CN116554505 A CN 116554505A CN 202310688050 A CN202310688050 A CN 202310688050A CN 116554505 A CN116554505 A CN 116554505A
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- Prior art keywords
- maca polysaccharide
- solution
- aqueous solution
- chitosan
- hydrogel
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Abstract
本发明公开一种玛咖多糖水凝胶的制备方法及其应用，将醛基玛咖多糖、N‑羧乙基壳聚糖的水溶液按比例混合并加入银纳米粒子，充分搅拌得到多糖水凝胶，该水凝胶具有均匀的多孔结构，流变学结果显示出固态弹性特征；本发明制备方法简单，通过席夫碱反应制得，避免了有毒交联剂的使用，制备过程安全、易操作，而且原料易得、成本低。
Description
技术领域
本发明属于生物技术领域，尤其涉及一种玛咖多糖水凝胶的制备方法及其应用。
背景技术
日常生活中发生的意外事故比如烫伤、车祸、摩擦等造成皮肤的损伤。创面愈合过程漫长且存在被细菌感染化脓的风险。水凝胶是一种三维含水的聚合物网络，能够维持伤口的湿润度并保持伤口的通透性，减少出血抑制细菌的产生，在创面愈合过程中有着积极的影响。但现有的使用的一些化学交联剂有一定的细胞毒性，不建议使用。天然多糖制备水凝胶避免了有毒交联剂的使用，所以多糖水凝胶是绿色、经济、安全的产品。
玛咖多糖是中药玛咖的主要活性成分，含有发挥抗炎、抗氧化、免疫调节等多种药理作用的关键成分，且玛咖多糖表现出促进细胞增殖的活性。相对于合成的高分子材料具有个更高的生物安全性，更容易满足生理需求，但较差的力学性能和流动性又限制了其的应用。借助玛咖多糖的稳定性和主链结构上的羟基易修饰性，有望通过修饰后与其他大分子共同设计出一种创新的生物材料，并保持原有药理性能，开拓其在药理活性、药物传递等领域的应用。
壳聚糖是甲壳素脱去乙酰基的产物，甲壳素广泛存在于虾蟹等甲壳类动物的壳中，是地球上仅次于植物纤维的第二大生物资源，也是自然界中唯一存在的碱性多糖。其具有降血脂、抗菌消炎、促进伤口愈合的作用。但它不溶于水和大多数有机溶剂，只溶于稀酸溶液，因此限制了它在药用材料中的应用。因此可以对其进行修饰，增加其在水中是溶解度，开拓其应该范围。
银纳米粒子是一种具有抗菌活性的纳米材料，目前在日常生活中已被广泛应用，银纳米粒子能够通过多种机制起到杀菌作用而不易产生耐药性。当银纳米粒子粒径减小时，其比表面积增大，抗菌活性能够增强。但高溶度的银分布会对细胞、组织和器官造成明确的毒性和损伤。因此需要严格控制其在伤口部位的浓度。
发明内容
本发明提供一种玛咖多糖水凝胶的制备方法及其应用，以中药玛咖中活性成分玛咖多糖与天然材料壳聚糖为基质材料，经改性后，再与银纳米粒子一起，通过席夫碱反应制备水凝胶，此水凝胶具有很好的力学性能和优异的抗菌活性以及作为药物载体的潜力，同时水凝胶是一种三维的聚合物网络，可以缓慢的释放其中的药物，避免了药物在伤口部位的蓄积，本发明玛咖多糖水凝胶可作为伤口愈合过程中潜在的修复材料。
本发明的技术方案如下：
一种玛咖多糖水凝胶的制备方法，主要包括如下步骤：
(1)对玛咖多糖进行氧化反应，获得醛基玛咖多糖；
(2)对壳聚糖进行迈克尔加成反应，获得水溶性N-羧乙基壳聚糖；
(3)对硝酸银进行还原反应，获得银纳米粒子；
(4)将醛基玛咖多糖溶于水中，配制成醛基玛咖多糖水溶液；将N-羧乙基壳聚糖溶于水中，配制成N-羧乙基壳聚糖水溶液并将步骤(3)的银纳米粒子加入其中，得到含银纳米粒子的N-羧乙基壳聚糖水溶液；
(5)将醛基玛咖多糖水溶液与含银纳米粒子的N-羧乙基壳聚糖水溶液混合，静置5-10min，醛基玛咖多糖的醛基和N-羧乙基壳聚糖的氨基发生席夫碱反应，胶凝形成，制得含银纳米粒的玛咖多糖-壳聚糖水凝胶。
步骤(1)中所述氧化反应为高选择性氧化剂高碘酸钠氧化玛咖多糖中的羟基，生成醛基玛咖多糖，氧化反应具体步骤如下：
(1)将0.5-1.5g玛咖多糖溶于50mL水中，配制成玛咖多糖水溶液；
(2)向步骤(1)的玛咖多糖水溶液中加入132-460mg高碘酸钠，在室温、避光、氮气保护条件下进行氧化反应12-48h，向反应体系内加入反应终止剂乙二醇0.2-0.8mL以消耗未反应完全的高碘酸钠，对氧化反应后的溶液进行透析处理，透析使用截留分子量为3500Da的透析袋，每天更换三次水，透析时间为三天，对透析产物进行冷冻干燥处理，得到醛基玛咖多糖。
步骤(2)中所述迈克尔加成反应按照如下步骤进行：
(1)将2-6g壳聚糖溶于200mL水中，形成壳聚糖水溶液；
(2)向步骤(1)的壳聚糖水溶液中加入5-6mL丙烯酸，并将其在40-60℃下进行迈克尔加成反应1-3天；
(3)用浓度为1mol/L氢氧化钠溶液对步骤(2)的迈克尔加成反应后的溶液调节pH＝10-12，对调节pH后的溶液进行透析处理，透析使用截留分子量为3500Da的透析袋，每天更换三次水，透析时间为三天，对透析产物进行冷冻干燥处理，得到N-羧乙基壳聚糖。
步骤(3)中所述还原反应按照如下步骤进行：
(1)在避光条件下，将30-50mg聚乙烯吡咯烷酮加入到20mL水中并加热至沸腾；
(2)向步骤(1)沸腾的溶液中加入0.3-0.8mL浓度为0.1mol/L的硝酸银溶液、5-30μL浓度为1mol/L的氢氧化钠溶液；
(3)向步骤(2)的溶液中加入0.3-0.5mL浓度为0.1mol/L的柠檬酸钠溶液，搅拌1h，冷却至室温后，对还原反应后的溶液进行离心处理，并对离心产物进行冷冻干燥处理，得银纳米粒子。
步骤(4)所述醛基玛咖多糖水溶液的质量浓度为1-3％；所述的含银纳米粒子的N-羧乙基壳聚糖水溶液中N-羧乙基壳聚糖的质量浓度为1-3％，银纳米粒子的质量浓度为0.002-0.01％。
步骤(5)中所述醛基玛咖多糖水溶液与含银纳米粒子的N-羧乙基壳聚糖水溶液混合的体积比为1:(1-3)。
本发明还提供所述玛咖多糖水凝胶作为药用材料应用于抑制细菌生长、创伤修复。
与现有技术相比，本发明具有如下明显优点：
本发明通过席夫碱反应制备动态交联水凝胶，在凝胶制备过程中避免了有机试剂的使用，使得水凝胶具有良好的生物安全性。
本发明在水凝胶中引用银纳米粒子，使水凝胶具有更好的抗菌活性。
本发明中药活性成分玛咖多糖和天然生物材料壳聚糖经改性修饰处理，使其成为制备水凝胶的基质材料，其中玛咖多糖发挥“药辅双效”的作用，既作为基质参与水凝胶的构建，又表现出良好的促进愈合功能。且本发明中使用的玛咖多糖、壳聚糖便宜易得，避免了高成本原料的使用，成本较低。
附图说明
图1为MP、MP-CHO的傅里叶红外变换光谱图，其中MP：玛咖多糖；MP-CHO：醛基玛咖多糖；
图2为CS、CECS的傅里叶红外变换光谱图，其中CS：壳聚糖；CECS：N-羧乙基壳聚糖；
图3为CS、CECS的核磁共振氢谱；
图4为银纳米粒子的紫外吸收光谱图；
图5为银纳米粒子的透射电镜图；
图6为MP-CHO、CECS和MP-gel的傅里叶红外变换光谱图，其中MP-gel：玛咖多糖水凝胶；
图7为玛咖多糖水凝胶的扫描电镜图；
图8为玛咖多糖水凝胶的流变学时间扫描图；
图9为玛咖多糖水凝胶的流变学振荡频率图；
图10为玛咖多糖水凝胶的溶胀率图；
图11为玛咖多糖水凝胶的溶血率图，其中PBS：磷酸盐缓冲液；Trition X-100：曲拉通；
图12为玛咖多糖水凝胶的抑菌实验图，其中S.auerus：金黄色葡萄球菌；E.coli：大肠杆菌；
图13为玛咖多糖水凝胶对人成纤维细胞的迁移图；
图14为玛咖多糖水凝胶对人成纤维细胞的迁移率图；
图15为玛咖多糖水凝胶对大鼠刺激性和组织病理切片图。
具体实施方式
下面结合附图并通过具体实施例来进一步详细说明本发明，但本发明并不局限于这些实施例。
本发明中使用的玛咖多糖由中药玛咖中提取得到，其提取方法参照：Guo,TingtingYang,YeGao,MingjuQu,YuanGuo,XiaoxiLiu,YuanCui,XiumingWang,Chengxiao.LepidiummeyeniiWalpers polysaccharide and its cationic derivative re-educatetumor-associated macrophages for synergistic tumor immunotherapy[J].Carbohydrate Polymers:Scientific and Technological Aspects of IndustriallyImportant Polysaccharides,2020,250(1)。
其他如无特别说明，本发明的实施例中的原料均通过商业途径购买。
实施例1
制备醛基玛咖多糖(MP-CHO)，具体步骤如下：
(1)配制玛咖多糖水溶液
准确称取1g玛咖多糖(MP)加入到50mL去离子水中，室温下搅拌1h使其充分溶解，配制成玛咖多糖水溶液，其中玛咖多糖的质量浓度为2％；
(2)高碘酸钠的加入
称取高碘酸钠粉末263mg，加入到步骤(1)的玛咖多糖水溶液中，并混合均匀，配制成含有高碘酸钠的多糖水溶液；
(3)氧化反应
步骤(2)的混合溶液在室温、避光、氮气保护条件下，高碘酸钠与玛咖多糖发生氧化反应，将玛咖多糖中邻位羟基氧化成醛基，生成醛基玛咖多糖(MP-CHO)，氧化反应24h后，加入反应终止剂乙二醇0.5mL，继续搅拌1h，以消耗剩余的高碘酸钠终止氧化反应；
(4)透析处理
将步骤(3)反应后的溶液装入截留分子量为3500Da透析袋中，在去离子水中进行透析处理，每天更换三次水(每8小时更换一次水)，透析时间为三天，透析结束后将透析产物于-20℃冰箱预冻后，于冷冻真空干燥机中进行冷冻干燥，得到醛基玛咖多糖(MP-CHO)。
采用盐酸羟胺滴定法测定步骤(4)制备的醛基玛咖多糖的氧化度，制得的醛基玛咖多糖(MP-CHO)的氧化度为18.2％。
取玛咖多糖和实施例1步骤(4)制得的醛基玛咖多糖，分别与溴化钾研磨混合压片，以空白溴化钾片作为对照，设定波数范围为4000-400cm-1，在傅里叶变换红外光谱仪上对样品的红外光谱进行测定；红外光谱测定结果如图1所示，由图1可知，在3440cm-1处为O-H的伸缩振动，2925cm-1处代表糖类-CH3或者-CH2中C-H伸缩振动，在1163cm-1和1024cm-1处为吡喃糖的特征吸收峰，与玛咖多糖相比，醛基玛咖多糖在1735cm-1处新吸收峰代表醛基C＝O的伸缩振动，红外图谱结果证实了玛咖多糖被氧化为醛基玛咖多糖。
实施例1中的玛咖多糖的添加量在0.5-1.5g波动，玛咖多糖水溶液的质量浓度在1-3％波动，高碘酸钠的添加量在132-460mg波动，其质量浓度在0.264-0.92％波动，反应终止剂乙二醇0.2-0.8mL波动，最后得到的结果与实施例1相同。
实施例2
制备N-羧乙基壳聚糖(CECS)，具体步骤如下：
(1)配制壳聚糖水溶液
准确称取4g壳聚糖(CS)加入到200mL去离子水中，搅拌5min使其充分分散，配制成壳聚糖水溶液，其中壳聚糖的质量浓度为2％；
(2)丙烯酸的加入
量取5.84mL丙烯酸，加入到步骤(1)的壳聚糖水溶液中，并混合均匀，配制成含有丙烯酸的壳聚糖溶液；
(3)迈克尔加成反应
将步骤(2)的混合溶液置于50℃下，丙烯酸与壳聚糖上的胺基发生迈克尔加成反应，其中反应时间为2天，向冷却至室温的反应后溶液中加入浓度为1mol/L的氢氧化钠溶液，将溶液pH值调至11，制得所述N-羧乙基壳聚糖；
(4)透析处理
将步骤(3)反应后的溶液装入截留分子量为3500Da透析袋中，在去离子水中进行透析处理，每天更换三次水(每8小时更换一次水)，透析时间为三天，透析结束后将透析袋内的透析产物于-20℃冰箱预冻后，于冷冻真空干燥机中进行冷冻干燥，得到N-羧乙基壳聚糖(CECS)。
取壳聚糖原料和本实施例步骤(4)制得的N-羧乙基壳聚糖，分别与溴化钾研磨混合压片，以空白溴化钾片作为对照，设定波数范围为4000-400cm-1，在傅里叶变换红外光谱仪上对样品的红外光谱进行测定，红外光谱测定结果如图2所示，由图2可知，在3427cm-1处为O-H、N-H的伸缩振动的叠加，2920cm-1、2886cm-1处代表糖类-CH3或者-CH2中C-H伸缩振动，在1651cm-1处为酰基中C＝O伸缩振动，1594cm-1处为伯胺基N-H的弯曲振动，与壳聚糖相比N-羧乙基壳聚糖在1565cm-1处新的吸收峰为伯胺基的N-H弯曲振动与COO-的不对称伸缩振动的叠加，红外图谱结果证实了壳聚糖经迈克尔加成反应生成N-羧乙基壳聚糖。
取壳聚糖原料和本实施例步骤(4)制得的N-羧乙基壳聚糖，分别溶于体积百分比为1％的CD3COOD的D2O溶液，在核磁共振波谱仪上对样品进行氢谱扫描测定，核磁共振波谱的结果如图3所示，由图3可知，壳聚糖在1.8ppm出的化学位移为壳聚糖中未脱掉的N-乙酰的-CH3残基化学位移峰，在2.3ppm和3.25ppm处出现两个新峰，表明存在亚甲基质子，说明通过迈克尔加成反应可以成功地将丙烯酸接枝到壳聚糖的胺基上，制得的N-羧乙基壳聚糖(CECS)的取代度由公式：取代度(DS)＝(1-DD)×3/2×A2.3/A1.8×100％计算可得为29.4％，其中壳聚糖是脱乙酰度(DD)为93％。
实施例2中的壳聚糖的添加量在2-6g波动，壳聚糖水溶液的质量浓度在1-3％波动，丙烯酸的添加量在5-6mL波动，反应温度在40-60℃波动，反应时间在1-3天波动，反应后的溶液调节的pH值在10-12波动，最后得到的结果与实施例2相同。
实施例3
制备银纳米粒子，具体步骤如下：
(1)配制聚乙烯吡咯烷酮水溶液
准确称取40mg聚乙烯吡咯烷酮加入到20mL去离子水中，搅拌5min使其充分溶解，在避光下加热至沸腾，配制成聚乙烯吡咯烷酮水溶液，其中聚乙烯吡咯烷酮的质量浓度为0.2％；
(2)硝酸银溶液和氢氧化钠溶液的加入
量取0.6mL浓度为0.1mol/L硝酸银溶液、10μL浓度为1mol/L氢氧化钠溶液，依次加入到步骤(1)的聚乙烯吡咯烷酮水溶液中，并混合均匀；
(3)柠檬酸钠溶液的加入
量取0.4mL浓度为0.1mol/L柠檬酸钠溶液，加入到步骤(2)的溶液中，并混合均匀；
(4)还原反应
柠檬酸钠与硝酸银发生还原反应，将硝酸银还原成银纳米粒子，搅拌还原时间为1h；
(5)离心处理
将步骤(4)反应后的溶液冷却至室温后，在12000r/min离心10min，将离心后的沉淀物于冷冻真空干燥机中进行冷冻干燥，得到银纳米粒子。
取步骤(5)制得的银纳米粒子，溶于去离子水中，在紫外分光光度计上对样品进行全波长扫描，扫描结果如图4所示，由图4可知，在440nm处的特征吸收峰，且其他波长处无其他吸收峰，表明银纳米粒子的存在。
取步骤(5)制得的银纳米粒子，溶于去离子水中，在透射电镜仪上对银纳米粒子的形貌就行观察，透射电镜结果如图5所示，由图5可知，银纳米粒子成均匀分布，粒径在35-40nm，表明银纳米粒子成均匀稳定状态的存在。
实施例3的聚乙烯吡咯烷酮的添加量在30-50mg波动，硝酸银溶液的添加量在0.3-0.8mL波动，氢氧化钠溶液在5-30μL波动，柠檬酸钠溶液在0.3-0.5mL波动，最后得到的结果与实施例3同。
实施例4
制备玛咖多糖水凝胶(MP-gel)，具体步骤如下：
(1)将实施例1制备的醛基玛咖多糖溶于水，配置成醛基玛咖多糖水溶液，其中醛基玛咖多糖水溶液的质量百分比浓度为2％；
(2)将实施例2制备的N-羧乙基壳聚糖溶于水，配置成N-羧乙基壳聚糖水溶液，并将实施例3制备的银纳米子加到N-羧乙基壳聚糖水溶液中，其中N-羧乙基壳聚糖水溶液的质量百分比浓度为2％，银纳米粒子的质量百分比浓度为0.005％；
(3)将醛基玛咖多糖水溶液与含银纳米粒子的N-羧乙基壳聚糖水溶液混合，并于室温下静置，进行胶凝处理8min，醛基玛咖多糖水溶液和含银纳米粒子的N-羧乙基壳聚糖水溶液的体积之比为1:2，得到玛咖多糖水凝胶(MP-gel)。
实施例5
制备玛咖多糖水凝胶(MP-gel)，具体步骤如下：
(1)将实施例1制备的醛基玛咖多糖溶于水，配置成醛基玛咖多糖水溶液，其中醛基玛咖多糖水溶液的质量百分比浓度为1％；
(2)将实施例2制备的N-羧乙基壳聚糖溶于水，配置成N-羧乙基壳聚糖水溶液，并将实施例3制备的银纳米子加到N-羧乙基壳聚糖水溶液中，其中N-羧乙基壳聚糖水溶液的质量百分比浓度为3％，银纳米粒子的质量百分比浓度为0.002％；
(3)将醛基玛咖多糖水溶液与含银纳米粒子的N-羧乙基壳聚糖水溶液混合，并于室温下静置，进行胶凝处理5min，醛基玛咖多糖水溶液和含银纳米粒子的N-羧乙基壳聚糖水溶液的体积之比为1:3，得到玛咖多糖水凝胶(MP-gel)。
实施例6
制备玛咖多糖水凝胶(MP-gel)，具体步骤如下：
(1)将实施例1制备的醛基玛咖多糖溶于水，配置成醛基玛咖多糖水溶液，其中醛基玛咖多糖水溶液的质量百分比浓度为3％；
(2)将实施例2制备的N-羧乙基壳聚糖溶于水，配置成N-羧乙基壳聚糖水溶液，并将实施例3制备的银纳米子加到N-羧乙基壳聚糖水溶液中，其中N-羧乙基壳聚糖水溶液的质量百分比浓度为1％，银纳米粒子的质量百分比浓度为0.01％；
(3)将醛基玛咖多糖水溶液与含银纳米粒子的N-羧乙基壳聚糖水溶液混合，并于室温下静置，进行胶凝处理10min，醛基玛咖多糖水溶液和含银纳米粒子的N-羧乙基壳聚糖水溶液的体积之比为1:1，得到玛咖多糖水凝胶(MP-gel)。
性能检测：
1、红外光谱实验
将实施例4中制备的玛咖多糖水凝胶进行冷冻干燥，将干燥后的MP-gel与溴化钾研磨混合压片，以空白溴化钾片作为对照，设定波数范围为4000-400cm-1，在傅里叶变换红外光谱仪上对样品的红外光谱进行测定。
红外光谱测定结果如图6所示，由图6可知，与MP-CHO的红外光谱图相比在1735cm-1处的醛基C＝O有关的伸缩振动峰消失，1651cm-1处出现的新峰为C＝N的伸缩振动峰，特征峰的变化表明醛基玛咖多糖的醛基与N-羧乙基壳聚糖的胺基发生了席夫碱反应，醛基特征峰基本消失，形成了新的C＝N键峰，证明玛咖多糖水凝胶的生成。
2、冻干水凝胶的扫描电镜实验
将实施例4中制备的玛咖多糖水凝胶进行冷冻干燥，随后将其浸泡在液氮中脆断，将脆断面放置在离蒸发源10-15cm处的样品台上进行旋转活动，喷金，完成后观察水凝胶内部形貌。
扫描电镜测定结果如图7所示，由图7可知，水凝胶内部呈现多孔结构，连续性好，这种优异三维空间结构，有利于水分子的自由扩散，其牢固的骨架结构，使得水凝胶能吸收大量水分，溶胀而不破裂；若将该复合水凝胶用于伤口敷料，可以与创面紧密贴合，并提供一个良好的湿润微环境，还能够吸收伤口渗出液，防止感染，促进创面愈合。同时，水凝胶的孔隙结构可以作为药物的天然储库达到缓慢释放药物有效增加药物的生物利用度。
3、玛咖多糖水凝胶的流变学实验
将实施例4中制备的玛咖多糖水凝胶进行流变测试，采用平行板直径40mm、间隙1mm的流变仪测定了水凝胶的流变学特性，测定了在时间扫描模式和振荡频率模式下，水凝胶的存储模量(G')和损耗模量(G”)：
(a)时间扫描试验在室温下进行，恒定频率为1.59Hz，应变为1％；
(b)振荡频率试验在室温下进行，剪切率设置为0.1rad/s～100rad/s，恒定应变为1％。
流变实验结果如图8、9所示，由图8可知，G'和G”在开始时都很低，G'小于G”时，与系统的溶胶状态相对应，随着时间的推移，由于在醛基玛咖多糖和N-羧乙基壳聚糖之间形成了席夫碱键，G'和G”都增加了，G'的增长速度快于G”，不同的生长速率导致了凝胶化点(tgel)的出现，随着醛基玛咖多糖与N-羧乙基壳聚糖的持续交联，G'和G”持续增加，但G'的生长速度明显快于G”，这意味着水凝胶强度的增长，由图9可知，存储模量和损耗模量随频率而增加，这意味着理想的弹性和固态可以使凝胶成为伤口敷料的合格候选材料。
4、玛咖多糖水凝胶的溶胀实验
将实施例4中制备的玛咖多糖水凝胶进行冷冻干燥，取冻干水凝胶置于37℃磷酸盐缓冲液(模拟体液)中，每隔一小时取出水凝胶并用吸水纸吸干外层水，测量其质量，直至质量不在上升。溶胀结果如图10所示，由图10可知，随着时间的延长水凝胶不断的吸收外部的水，由溶胀率(％)＝(Wt-W0)/Wt×100％，计算可知在3h左右溶胀率达到平衡，溶胀率为1900％左右，表明水凝胶具有优良的吸水性能。
5、玛咖多糖水凝胶的溶血实验
将实施例4中制备的1g玛咖多糖水凝胶置于3mL磷酸盐缓冲液中，37℃浸泡24h，获得水凝胶浸提液，向水凝胶浸提液、阴性对照PBS、阳性对照质量浓度2％的Trition X-100中分别加入500μL的红细胞悬液，将上述溶液置于37℃下继续培养1h，1h后3500r/min离心5min，取上清，用紫外分光光度仪在490nm处测OD值；溶血率(％)＝(ODgel-OD阴)/(OD阳-OD阴)×100％，溶血结果如图11所示，由图11可知，水凝胶浸提液组基本上没有发生溶血，其溶血率为1.4％，低于5％的警戒线值，Trition X-100组发生很显的溶血现象，表明水凝胶具有很好的生物安全性。
6、玛咖多糖水凝胶的抑菌实验
将实施例4中制备的1g玛咖多糖水凝胶和1g磷酸盐缓冲液(空白组)分别与4mL的金黄色葡萄球菌和大肠杆菌细菌悬液在37℃孵育12h，将孵育后的菌液接种到的琼脂平板上，24h小时后观察菌落数，抑菌结果如图12所示，由图12可知，水凝胶组的琼脂平板上的菌落数要明显少于空白组，表明水凝胶对两种致病菌均具有良好的抗菌活性。
7、玛咖多糖水凝胶对人成纤维细胞(hacat)的迁移实验
细胞集体迁移被认为是反映伤口愈合过程的重要指标，将实施例4中制备的1g玛咖多糖水凝胶置于10mL完全培养基中，在37℃下浸泡24h，获得水凝胶浸提液，采用细胞划痕实验考察水凝胶浸提液对体外细胞迁移和增殖的影响，按照5×105/mL的密度将hacat细胞接种于6孔细胞培养板中，待细胞贴壁后吸弃旧培养基，使用无菌200μL枪头轻轻划过孔板底部产生细胞划痕，用PBS冲洗掉滑落的细胞，每孔加入2mL实施例4中制备的玛咖多糖水凝胶浸提液，空白组加入2mL完全培养基，分别于0h、6h、12h、24h取出培养板，观察细胞迁移情况，并用倒置显微镜拍照录，使用Image J软件对细胞迁移面积定量分析。
迁移结果如图13所示，由图13可知，各组细胞划痕面积随时间推移逐渐减小，与空白组相比水凝胶浸提液对细胞迁移具有更好的促进作用，水凝胶浸提液组的细胞在24h后出现划痕两侧接触的情况，划痕面积缩小程度较大。
由图14可知，迁移率(％)＝(T0-Th)/T0×100％，由Image J软件对细胞迁移面积的进行定量分析，浸提液处理组在24h后细胞迁移率接近90％，而空白处理组在43％左右，水凝胶浸提液对hacat有更高的促进生长迁移的作用，其中T0和Th为0h的划痕面积和预设的时间间隔的面积。
8、玛咖多糖水凝胶对大鼠皮肤的刺激性和生物安全性研究
健康的8周龄雄性SD(Sprague Dawley)大鼠(体重200-220g)，采用SD大鼠背侧全层皮肤模型评价实施例4中制备的玛咖多糖水凝胶对皮肤的刺激性研究，将SD大鼠麻醉后使用电动剃毛器将其背部毛发剃除，在其皮肤上涂抹水凝胶，空白组用PBS代替，每天涂抹，连续涂抹一周，一周后用皮肤美容仪对皮肤表面进行拍照并对涂抹处组织进行病理切片观察。
由图15可知，水凝胶处理组的大鼠皮肤表面没有红肿溃烂现象的出现，由组织病理切片可知，水凝胶涂抹部位组织胶原蛋白的分布均匀未观察到明显的刺激或炎症，表明本发明水凝胶对皮肤比较温且无刺激性，适用于创面修复。
综上所述，本发明制备玛咖多糖水凝胶，操作简单，经济易得，水凝胶具良好的抗菌活性和促进细胞迁移的能力，水凝胶还可以作为药物载体有效减缓药物释放过程，本发明的玛咖多糖水凝胶可以促进创面修复，加速创伤愈合，在临床外科应用中具有广阔前景。
Claims (7)
1.一种玛咖多糖水凝胶的制备方法，其特征在于，包括以下步骤：
(1)玛咖多糖进行氧化反应，制备获得醛基玛咖多糖；
(2)壳聚糖进行迈克尔加成反应，制备获得N-羧乙基壳聚糖；
(3)硝酸银进行还原反应，制备获得银纳米粒子；
(4)将醛基玛咖多糖溶于水中，配制成醛基玛咖多糖水溶液；将N-羧乙基壳聚糖溶于水中，配制成N-羧乙基壳聚糖水溶液并将步骤(3)的银纳米粒子加入其中，得到含银纳米粒子的N-羧乙基壳聚糖水溶液；
(5)将醛基玛咖多糖水溶液与含银纳米粒子的N-羧乙基壳聚糖水溶液混合，静置5-10min，制得水凝胶。
2.根据权利要求1所述的制备方法，其特征在于，步骤(1)中所述氧化反应具体步骤如下：
(1)将0.5-1.5g玛咖多糖溶于50mL水中，配制成玛咖多糖水溶液；
(2)向步骤(1)玛咖多糖水溶液中加入132-460mg高碘酸钠，在室温、避光、氮气保护条件下进行氧化反应12-48h，向反应体系内加入反应终止剂乙二醇0.2-0.8mL，对氧化反应后的溶液使用3500Da的透析袋进行透析处理，每天更换三次水，透析三天，透析产物进行冷冻干燥处理，得到醛基玛咖多糖。
3.根据权利要求1所述的制备方法，其特征在于，步骤(2)中所述迈克尔加成反应按照如下步骤进行：
(1)将2-6g壳聚糖溶于200mL水中，形成壳聚糖水溶液；
(2)向步骤(2)的壳聚糖水溶液中加入5-6mL丙烯酸，在40-60℃下进行迈克尔加成反应1-3天；
(3)用浓度为1mol/L氢氧化钠溶液将步骤(2)反应后的溶液调节pH＝10-12，然后使用3500Da的透析袋对反应液进行透析处理，每天更换三次水，透析时间为三天，对透析产物进行冷冻干燥处理，得到N-羧乙基壳聚糖。
4.根据权利要求1所述的制备方法，其特征在于，步骤(3)中所述还原反应按照如下步骤进行：
(1)在避光条件下，将30-50mg聚乙烯吡咯烷酮加入到20mL水中并加热至沸腾；
(2)向步骤(1)沸腾的溶液中加入0.3-0.8mL浓度为0.1mol/L的硝酸银溶液、5-30μL浓度为1mol/L的氢氧化钠溶液，混匀；
(3)向步骤(2)溶液中加入0.3-0.5mL浓度为0.1mol/L的柠檬酸钠溶液，搅拌1h，冷却至室温后，对反应后的溶液进行离心处理，对离心产物进行冷冻干燥处理，得到银纳米粒子。
5.根据权利要求1所述的制备方法，其特征在于，步骤(4)醛基玛咖多糖水溶液的质量浓度为1-3％；所述含银纳米粒子的N-羧乙基壳聚糖水溶液中N-羧乙基壳聚糖的质量浓度为1-3％，银纳米粒子的质量浓度为0.002-0.01％。
6.根据权利要求1所述的制备方法，其特征在于，步骤(5)中醛基玛咖多糖水溶液与含银纳米粒子的N-羧乙基壳聚糖水溶液混合的体积比为1:1-3。
7.权利要求1所述方法制备得到的玛咖多糖水凝胶作为创面修复材料应用于抑制细菌生长、创伤修复。
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