CN113956413A - 一种纳米复合水凝胶的制备方法及其在促糖尿病伤口愈合中的应用 - Google Patents
一种纳米复合水凝胶的制备方法及其在促糖尿病伤口愈合中的应用 Download PDFInfo
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- CN113956413A CN113956413A CN202111335986.4A CN202111335986A CN113956413A CN 113956413 A CN113956413 A CN 113956413A CN 202111335986 A CN202111335986 A CN 202111335986A CN 113956413 A CN113956413 A CN 113956413A
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Abstract
本专利提供了一种纳米复合水凝胶的制备方法及其在促糖尿病伤口愈合中的应用。所述方法包括：制备功能化改性的天然高分子聚合物与功能金属氧化物纳米颗粒，将功能化改性的天然高分子聚合物溶液与功能金属氧化物纳米颗粒混合，最后通过酶促交联和光交联的手段制备纳米复合水凝胶。由该方法制备的水凝胶具有多重功能，可用于调节糖尿病创面的病理微环境以促进其愈合。
Description
技术领域
本发明属于组织工程与高分子材料领域，具体涉及一种纳米复合水凝胶的制备方法及其在促糖尿病伤口愈合中的应用。
背景技术
本发明公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
慢性疾病，包括糖尿病、心脑血管疾病、缺氧、癌症和免疫抑制等都会引发慢性伤口的形成，而慢性伤口不能正常、有序、及时地修复，这给患者和医疗系统都造成了极大的负担。其中，糖尿病创面在愈合过程中会发生过度的氧化应激反应，同时分泌大量的ROS，导致伤口经历长时间的炎症浸润，相关免疫细胞功能失调，影响血管和组织生成。同时，血管生成受阻大大降低了伤口的O2和营养物质的递送效率，进一步延长炎症期，这些因素都使糖尿病伤口不遵循正常伤口愈合阶段的线性发展，导致出现慢性非愈合伤口。
目前，临床治疗糖尿病伤口的主要方法为：覆盖纱布等敷料、施用药物、皮瓣修复等，新兴的治疗手段还包括使用高压氧疗法通过增加局部含氧量来改善伤口微环境，使用负压辅助伤口疗法改善局部血液供应，以及使用富含细胞因子和营养因素的富含血小板的血浆来治疗慢性伤口。然而，这些方法存在药物副作用、递送效率低、需要麻醉、免疫排斥等问题，简而言之，上述所有治疗方法均未能改变慢性伤口的微观环境，不利于修复，在慢性伤口的治疗上也未见突破，因此，亟需一种高效、低免疫原性的新策略用于糖尿病伤口愈合。
发明内容
为了解决上述技术的问题，本发明提供了一种纳米复合水凝胶的制备方法及其在促糖尿病伤口愈合中的应用，通过功能性纳米材料与凝胶支架结合改善糖尿病伤口的炎症微环境，加速伤口愈合。同时，水凝胶还具有可协调的机械性能，较好的生物相容性，且反应条件温和可控、有原位注射成型的潜力，在组织工程等领域具有极大的应用前景。本发明通过如下的技术方案来实现：
(1)对天然高分子材料1和2进行双键改性制备出聚合物A及B；
(2)制备功能金属氧化物纳米颗粒；
(3)将改性的聚合物A溶解在特异性酶溶液中制得溶液一；
(4)将改性的聚合物B溶解在光引发剂中，与步骤(2)制得的溶液一混合，得到溶液二；
(5)将功能氧化纳米颗粒均匀分散到步骤(4)制得的溶液二中得到溶液三；
(6)静置一段时间使酶促反应完全后，采用紫外光照射步骤(5)制得的溶液三，引发自由基聚合，使聚合物A与B的高分子链之间形成三维互穿网络，制得纳米复合水凝胶。
其中，步骤(1)所述聚合物A及B由以下方法制得：
将天然高分子材料1加入去离子水中，搅拌以完全溶解，
优选的，然高分子材料1为丝素蛋白、明胶，其水溶液的浓度为5～20g/L。
进一步的，加入待接枝物，继续反应2～12h。
优选的，待接枝的反应物为甲基丙烯酸缩水甘油酯、甲基丙烯酸酐、丙烯酸甲酯，高分子材料1与接枝物的摩尔比为1∶0.5～2，体系反应的温度为40-60℃。
进一步的，将完成的反应液置于去离子水中透析。
优选的，透析时间为2-7天。
进一步的，将透析过的溶液冷冻干燥即得到所述的改性高分子聚合物A。
将天然高分子材料2加入去离子水中，搅拌以完全溶解，
优选的，然高分子材料2为羧甲基纤维素、壳聚糖、透明质酸，其水溶液的浓度为10～20g/L。
进一步的，加入待接枝物，继续反应5～12h。
优选的，待接枝的反应物为甲基丙烯酸缩水甘油酯、甲基丙烯酸酐、丙烯酸甲酯，高分子材料1与接枝物的摩尔比为1∶0.5～2，体系反应的温度为60℃。
进一步的，将完成的反应液置于去离子水中透析。
优选的，透析时间为2-7天。
进一步的，将透析过的溶液冷冻干燥即得到所述的改性高分子聚合物B。
其中，步骤(3)所述的溶液一由如下方法制得：
用缓冲液溶解酶，配制成对应的酶溶液，之后将步骤(1)制得的改性高分子聚合物A溶解在配置好的酶溶液中，制得溶液一。
优选的，缓冲液为PBS缓冲液，酶为谷氨酰胺转氨酶，酶溶液浓度为0.16～0.8 g/L。
进一步的，将改性高分子聚合物A溶解在配置好的酶溶液中溶解。
优选的，溶液的浓度为50～150g/L。
其中，步骤(4)所述的溶液二由如下方法制得：
将步骤(1)制得的改性高分子聚合物B溶解于光引发剂溶液中，再将其与步骤(3)制得的溶液一混合得到溶液二。
优选的，光引发剂溶液的浓度为5～20g/L，改性高分子聚合物B的浓度为 50～100g/L。
其中，步骤(5)所述的溶液三由如下方法制得：
将纳米粒子均匀分散在步骤(4)制得的溶液二中，获得溶液三。
优选的，纳米粒子为二氧化铈纳米颗粒或二氧化锰纳米颗粒，纳米颗粒的浓度为0.5～5g/L。
进一步的，通过超声将纳米粒子均匀分散在体系中，制得溶液三。
其中，步骤(6)所述纳米复合水凝胶由如下方法制得：
将步骤(5)制得的溶液三进行酶促反应，构建一层网络后，于紫外光照射下，引发自由基聚合，形成稳固的第二层网络，最终聚合成胶。
优选的，紫外光的波长为365nm，光强度为10-300mW/cm2，照射时间为 0.5min～30min。
本发明还提供由上述方法制得的纳米复合水凝胶在组织工程材料领域中的应用。
所述应用包括但不限于：制备医用伤口敷料和细胞支架。
本发明的有益效果在于：
本发明引入生物相容性高、吸水能力优异的生物材料改性聚氨基酸和聚多糖为主体材料，通过酶促反应构建一层网络，再通过双键聚合高分子链构建双层交联网络，并将功能纳米粒子均匀分散在支架中，利用自由基聚合反应原理快速成胶。酶促反应选择性强，条件温和，紫外光引发的双键自由基聚合反应快速，条件温和可控。功能纳米粒子能有效缓解糖尿病伤口的氧化应激微环境。同时，聚氨基酸具有与天然蛋白质相似的二级结构，可模拟组织细胞基质中的蛋白成分仿生构建组织工程多孔支架，若作为组织工程材料，能够有效促进损伤后组织的再生与重建，另外，针对糖尿病伤口病理环境中过度表达的基质金属蛋白酶有很好的消耗作用。且凝胶具有可注射能力以及可调的机械性能、可用作定制化伤口敷料，且生物相容性好、操作条件温和简便等优点，在医用伤口敷料、细胞支架等生物医疗领域具有广阔的市场应用前景。
附图说明
图1是本发明所述改性丝素蛋白及改性羧甲基纤维素的机理图。
图2是本发明所述改性丝素蛋白的1H-NMR谱图。
图3是本发明所述改性羧甲基纤维素的1H-NMR谱图。
图4是本发明所述水凝胶纳米复合水凝胶的孔径图片(右图为局部放大图)。
图5是本发明所述水凝胶的细胞相容性实验结果。
图6是本发明所述水凝胶胞内清除活性氧的实验结果。
图7是本发明所述水凝胶促进糖尿病小鼠伤口愈合的实验结果。
具体实施方式
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例中所描述的内容仅用于说明本发明，而不应当也不会限制权利要求书中所详述的本发明。
实施例1
一种纳米复合水凝胶的制备方法如下(材料改性方法如图1所示)：
(1)在去离子水中加入可溶性丝素蛋白(分子量为2000～10000道尔顿)粉末，制成10g/L的丝素蛋白水溶液，待完全溶解后，调节pH至4.5，在反应温度60℃条件下加入0.5当量的甲基丙烯酸缩水甘油酯(GMA)搅拌溶解，继续反应6h。反应结束后，将反应液移至透析袋中，于去离子水中透析3天。将透析后得到的纯化溶液冷冻干燥得到甲基丙烯酸缩水甘油酯修饰的丝素蛋白聚合物(SF-GMA)，将冻干后的样品溶解通过核磁共振氢谱仪进行表征，得到样品的1H-NMR谱图如图2所示。
(2)去离子水中加入羧甲基纤维素(分子量为90,000道尔顿)粉末，制成10g/L的羧甲基纤维素水溶液，待完全溶解后，调节pH至5.1，在反应温度60℃条件下加入1.5当量的甲基丙烯酸缩水甘油酯(GMA)搅拌溶解，继续反应6h。反应结束后，将反应液移至透析袋中，于去离子水中透析3天。将透析后得到的纯化溶液冷冻干燥得到甲基丙烯酸缩水甘油酯修饰的纤维素聚合物(CMC- GMA)，将冻干后的样品溶解通过核磁共振氢谱仪进行表征，得到样品的1H- NMR谱图如图3所示。
(3)将四甲基氢氧化铵五水合物溶解在过氧化氢溶液中，配置成浓度为110 g/L的溶液。将四水氯化锰溶解在RO水中，配置成浓度为60g/L的溶液。之后，将两者快速混合，搅拌过夜。反应完之后，高速离心10min，将所得黑色固体分别通过水和乙醇洗涤三次，将所得物烘干获得二氧化锰纳米颗粒。
(4)用PBS缓冲液溶解谷氨酰胺酶，配制浓度为0.4g/L的谷氨酰胺转氨酶溶液，之后将SF-GMA作为溶质，以制得的酶溶液为溶剂溶解得到浓度为100 g/L的溶液一。配制浓度为5g/L的光引发剂溶液，并以其作为溶剂溶解CMC- GMA配制成浓度为50g/L的溶液二。将溶液一与溶液二混合均匀，将不同浓度的二氧化锰纳米粒子均匀分散在体系中得到溶液三。
(5)将溶液三轻微震荡10min，待酶促反应完全，之后采用紫外光照射溶液三，紫外光的波长为365nm，强度为40mW/cm2，自由基聚合反应被激活，SF- GMA与CMC-GMA形成纳米复合水凝胶支架，紫外光照射30s完全固化后，制得水凝胶样品1。
实施例2
(1)在去离子水中加入可溶性丝素蛋白(分子量为2000～10000道尔顿)粉末，制成10g/L的丝素蛋白水溶液，待完全溶解后，调节pH至4.7，在反应温度60℃条件下加入1.0当量的甲基丙烯酸缩水甘油酯(GMA)搅拌溶解，继续反应6h。反应结束后，将反应液移至透析袋中，于去离子水中透析3天。将透析后得到的纯化溶液冷冻干燥得到甲基丙烯酸缩水甘油酯修饰的丝素蛋白聚合物(SF-GMA)。
(2)去离子水中加入羧甲基壳聚糖(羧基化度80％)粉末，制成20g/L的羧甲基壳聚糖水溶液，待完全溶解后，调节pH至5.0，在反应温度60℃条件下加入1当量的甲基丙烯酸缩水甘油酯(GMA)搅拌溶解，继续反应8h。反应结束后，将反应液移至透析袋中，于去离子水中透析3天。将透析后得到的纯化溶液冷冻干燥得到甲基丙烯酸缩水甘油酯修饰的纤维素聚合物(CS-GMA)。
(3)用PBS缓冲液溶解谷氨酰胺酶，配制浓度为0.4g/L的谷氨酰胺转氨酶溶液，之后将SF-GMA作为溶质，以制得的酶溶液为溶剂溶解得到浓度为100 g/L的溶液一。配制浓度为5g/L的光引发剂溶液，并以其作为溶剂溶解CS-GMA 配制成浓度为50g/L的溶液二。将溶液一与溶液二混合均匀，将二氧化锰纳米粒子均匀分散在体系中得到溶液三，纳米粒子浓度为1g/L。
(4)将溶液三轻微震荡10min，待酶促反应完全，之后采用紫外光照射溶液三，紫外光的波长为365nm，强度为20mW/cm2，自由基聚合反应被激活，SF- GMA与CS-GMA形成纳米复合水凝胶支架，紫外光照射45s完全固化后，制得水凝胶样品2。
实施例3
(1)在去离子水中加入明胶(分子量为50000～100000道尔顿)颗粒，40℃溶解制成50g/L的明胶水溶液，待完全溶解后，在反应温度50℃条件下加入1 当量的甲基丙烯酸酐(MA)搅拌溶解，继续反应2h。反应结束后，将反应液移至透析袋中，于去离子水中透析3天。将透析后得到的纯化溶液冷冻干燥得到甲基丙烯酸酐修饰的明胶聚合物(GelMA)。
(2)去离子水中加入羧甲基纤维素(分子量为90,000道尔顿)粉末，制成 20g/L的羧甲基纤维素水溶液，待完全溶解后，调节pH至5.0，在反应温度60℃条件下加入1.5当量的甲基丙烯酸缩水甘油酯(GMA)搅拌溶解，继续反应6h。反应结束后，将反应液移至透析袋中，于去离子水中透析3天。将透析后得到的纯化溶液冷冻干燥得到甲基丙烯酸缩水甘油酯修饰的纤维素聚合物(CMC- GMA)。
(3)用PBS缓冲液溶解谷氨酰胺酶，配制浓度为0.8g/L的谷氨酰胺转氨酶溶液，之后将GelMA作为溶质，以制得的酶溶液为溶剂溶解得到浓度为100 g/L的溶液一。配制浓度为5g/L的光引发剂溶液，并以其作为溶剂溶解CMC- GMA配制成浓度为100g/L的溶液二。将溶液一与溶液二混合均匀，将二氧化铈纳米粒子均匀分散在体系中得到溶液三，纳米粒子浓度为5g/L。
(4)将溶液三轻微震荡10min，待酶促反应完全，之后采用紫外光照射溶液三，紫外光的波长为365nm，强度为100mW/cm2，自由基聚合反应被激活， GelMA与CMC-GMA形成纳米复合水凝胶支架，紫外光照射15s完全固化后，制得水凝胶样品3。
实施例4
(1)在去离子水中加入明胶(分子量为50000～100000道尔顿)颗粒，40℃溶解制成100g/L的明胶水溶液，待完全溶解后，在反应温度50℃条件下加入1 当量的甲基丙烯酸酐(MA)搅拌溶解，继续反应2h。反应结束后，将反应液移至透析袋中，于去离子水中透析3天。将透析后得到的纯化溶液冷冻干燥得到甲基丙烯酸酐修饰的明胶聚合物(GelMA)。
(2)去离子水中加入透明质酸(分子量为100,000道尔顿)粉末，制成20 g/L的透明质酸水溶液，待完全溶解后，调节pH至5.4，在反应温度60℃条件下加入2当量的甲基丙烯酸缩水甘油酯(GMA)搅拌溶解，继续反应6h。反应结束后，将反应液移至透析袋中，于去离子水中透析3天。将透析后得到的纯化溶液冷冻干燥得到甲基丙烯酸缩水甘油酯修饰的透明质酸聚合物(HA-GMA)。
(3)用PBS缓冲液溶解谷氨酰胺酶，配制浓度为0.8g/L的谷氨酰胺转氨酶溶液，之后将GelMA作为溶质，以制得的酶溶液为溶剂溶解得到浓度为100 g/L的溶液一。配制浓度为5g/L的光引发剂溶液，并以其作为溶剂溶解CMC- GMA配制成浓度为100g/L的溶液二。将溶液一与溶液二混合均匀，将二氧化锰纳米粒子均匀分散在体系中得到溶液三，纳米粒子浓度为5g/L。
(4)将溶液三轻微震荡10min，待酶促反应完全，之后采用紫外光照射溶液三，紫外光的波长为365nm，强度为40mW/cm2，自由基聚合反应被激活，GelMA 与CMC-GMA形成纳米复合水凝胶支架，紫外光照射20s完全固化后，制得水凝胶样品4。
实施例5
以实施例1制备的纳米复合水凝胶进行一系列测试：
(1)纳米复合水凝胶的孔径扫描
将实施例1构建的水凝胶真空干燥后，通过扫描电子显微镜观察水凝胶的微观孔径结构，如图4所示，水凝胶呈现出有序排列的孔径，同时孔径较大，这有利于其用作伤口敷料时的透气性，也利于营养物质以及氧气的输送。
(2)纳米复合水凝胶的细胞毒性测试
在三天的时间中，通过MTT法测试纳米复合水凝胶对小鼠成纤维细胞(L929) 的细胞毒性大小，以评估纳米复合水凝胶的生物安全性。简而言之，将L929细胞按5×104/mL的密度接种在96孔板中并培养过夜以贴壁。然后用含有不同浓度二氧化锰的水凝胶(0、0.05％、0.1％、0.5％和1％)分别处理细胞，继续培养24 个小时后，加入MTT，继续培养4小时，最后用酶标仪评估细胞活力。如图5所示，当二氧化锰的浓度在0.5％以内时，体系保持与阳性对照组相似的细胞毒性，证实其的生物应用安全性。
(3)纳米复合水凝胶胞内清除活性氧测试
使用活性氧探针(DCFH-DA)检测细胞内活性氧水平。首先将密度为 5×104/mL的L929细胞接种于24孔板贴壁培养24h，实验组加入凝胶样品和 H2O2共孵育12h，阴性对照组仅更换培养基，H2O2对照组仅添加H2O2，SF/CMC 水凝胶组为不含有纳米粒子的复合水凝胶，SF/CMC@MnO2水凝胶组为含有纳米粒子的复合水凝胶，最后使用ROS检测试剂盒装载DCF荧光探针。用荧光显微镜观察细胞的荧光。如图6所示，实验结果表明，施用SF/CMC@MnO2水凝胶的细胞荧光强度有所降低，说明水凝胶有清除胞内活性氧的能力，有望通过降低局部活性氧的水平促进糖尿病伤口愈合。
(4)纳米复合水凝胶促进糖尿病伤口愈合效果测试
首先，构建糖尿病小鼠模型，将建模成功的小鼠分为三组，分别构建直径为 1cm的全层皮肤缺损伤口。接着，对Control组仅施用市售的透明敷料膜(3M， TegadermTM)，实验组一在伤口处施用SF/CMC水凝胶(不含有纳米粒子的复合水凝胶)，实验组二在伤口处施用SF/CMC@MnO2水凝胶(含有纳米粒子的复合水凝胶)，并在外部使用TegadermTM膜覆盖。将每只小鼠分笼饲养，在第0、3、 7、14天时观察不同组小鼠的伤口愈合效果。如图7所示，结果表面，在伤口处施用SF/CMC@MnO2水凝胶的糖尿病小鼠，愈合速度最快，效果最好，显示出水凝胶具有促进糖尿病伤口愈合的能力。
综上所述，本发明制备纳米复合水凝胶，操作简单，条件温和可控，水凝胶具有良好的活性氧清除性能、孔径特点和生物相容性，且具有良好的促进糖尿病伤口愈合的效果，在未来的临床应用方面有极大前景。
需要说明的是：以上所述仅为本发明优选的实验方案，并非用于限定本发明的权利范围；同以上描述，对于相关技术领域的专门人士应可明了及实施，因此其他未脱离本发明所解释的精神下所完成的等效改变或修饰，均应包含在申请专利范围中。
Claims (8)
1.一种纳米复合水凝胶的制备方法及其在促糖尿病伤口愈合中的应用，其特征在于，该方法包括以下步骤：
(1)对天然高分子材料1和2进行双键改性制备出聚合物A及B；
(2)制备功能金属氧化物纳米颗粒；
(3)将改性的聚合物A溶解在特异性酶溶液中制得溶液一；
(4)将改性的聚合物B溶解在光引发剂中，与步骤(3)制得的溶液一混合，得到溶液二；
(5)将功能氧化纳米颗粒均匀分散到步骤(4)制得的溶液二中得到溶液三；
(6)静置一段时间使酶促反应完全后，采用紫外光照射步骤(5)制得的溶液三，引发自由基聚合，使聚合物A与B的高分子链之间形成三维互穿网络，制得纳米复合水凝胶。
2.根据权利要求1所述的制备方法，其特征在于，步骤(1)包括以下步骤：
(1-1)将天然高分子材料1和2分别用去离子水溶解后，加入待接枝的反应物，继续反应12～48h，待反应结束后于去离子水中透析2～7天，经冷冻干燥即得到改性高分子聚合物A和B。
(1-2)优选的，天然高分子材料1包括但不限于丝素蛋白、明胶；天然高分子材料2包括但不限于羧甲基纤维素、壳聚糖、透明质酸；待接枝的反应物包括但不限于甲基丙烯酸缩水甘油酯、甲基丙烯酸酐、丙烯酸甲酯。
3.根据权利要求1所述的制备方法，其特征在于，步骤(1-1)所述的体系反应温度为40-60℃，反应的pH值为3～8。
4.根据权利要求1所述的制备方法，其特征在于，步骤(1-1)所述的反应物的水溶液浓度为5～20g/L，反应物与接枝物的摩尔比为1∶0.5～1∶2。
5.根据权利要求1所述的制备方法，其特征在于，步骤(2)中所述功能氧化纳米颗粒包括但不限于二氧化铈纳米颗粒、二氧化锰纳米颗粒。
6.根据权利要求1所述的制备方法，其特征在于，步骤(3)中所述酶包括但不限于谷氨酰胺转氨酶。
7.根据权利要求1所述的制备方法，其特征在于，步骤(6)中，在波长为365nm的紫外光照射下光引发聚合成胶，紫外光强度为10-300mW/cm2。
8.根据权利要求1～7所述的制备方法得到纳米复合水凝胶。
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