CN116199909B - 一种在酸性条件下通过网络收缩快速形成水凝胶的方法 - Google Patents
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Abstract
本发明公开了一种在酸性条件下通过网络收缩快速形成水凝胶的方法，包括以下步骤：(1)合成在酸性条件下具有α螺旋结构的二聚体多肽，配制多肽水溶液；(2)配制4‑arm‑PEG‑Mal水溶液；(3)分别量取多肽水溶液和4‑arm‑PEG‑Mal水溶液，调节pH值，定容，再将4‑arm‑PEG‑Mal水溶液滴加至多肽水溶液上部，并持续振荡，放置在摇床上振荡，得水凝胶溶液；(4)将水凝胶溶液调节至酸性，网络收缩，得多肽/PEG网络水凝胶。本发明采用生物相容性极好的材料，在酸性条件下收缩形成水凝胶，为水凝胶的制备提供了新的途径。
Description
技术领域
本发明涉及水凝胶制备技术领域，具体涉及一种在酸性条件下通过网络收缩快速形成水凝胶的方法。
背景技术
水凝胶是以水为分散介质的凝胶，是网状交联结构的水溶性高分子中引入一部分疏水基团和亲水残基，亲水残基与水分子结合，将水分子连接在网状内部，而疏水残基遇水膨胀的一种交联聚合物。作为一种具有三维高分子网络结构的材料，水凝胶性质柔软，但能保持一定的形状且能吸收大量的水。
目前水凝胶的制备方法一般有两种：一种是烯类单体在交联剂存在下聚合而成，称为化学凝胶；另一种是由水溶性聚合物直接交联得到，这种交联既可为共价键型也可为非共价键型。非共价键交联型即为物理凝胶，其中的聚合物链可通过不同的相互作用如氢键、疏水缔合、静电作用形成结合区。此外，也可使用辐射交联法制备水凝胶。
目前尚无一种可以从溶液中通过聚合物收缩得到水凝胶的方法，这种新方法将在各种领域中起到关键性的作用。
发明内容
为了解决上述技术问题，本发明的目的是提供一种在酸性条件下通过网络收缩快速形成水凝胶的方法，采用线性的C-Switcch-C多肽等生物相容性极好的材料，在酸性条件下收缩形成水凝胶，为水凝胶的制备提供了新的途径。
本发明解决上述技术问题的技术方案如下：提供一种在酸性条件下通过网络收缩快速形成水凝胶的方法，包括以下步骤：
(1)合成在酸性条件下具有α螺旋结构的二聚体多肽，然后配制成11-12mg/mL的多肽水溶液；
(2)采用4-arm-PEG-Mal配制浓度为15-16mg/mL的4-arm-PEG-Mal水溶液；
(3)分别量取步骤(1)所多肽水溶液和步骤(2)所得4-arm-PEG-Mal水溶液1.5mL，调节pH值至7.5，然后定容至2mL，再将4-arm-PEG-Mal水溶液滴加至多肽水溶液上部，并持续振荡，随后放置在摇床上振荡0.4-0.6h，得多肽/PEG网络水凝胶溶液；
(4)将步骤(3)所得多肽/PEG网络水凝胶溶液调节至酸性，线性多肽结构转变为具有α螺旋结构的二聚体多肽结构，网络收缩，得多肽/PEG网络水凝胶。
进一步，4-arm-PEG-Mal为四臂聚乙二醇马来酰亚胺。
进一步，在酸性条件下具有α螺旋结构的二聚体多肽为C-Switcch-C多肽。
进一步，步骤(1)中，C-Switcch-C多肽的氨基酸序列如SEQ ID NO.1所示，序列为：CENQSLEQENSQLKQEISQLEQEIQQLHYGC。
进一步，多肽采用的合成美国CEM Liberty Blue全自动多肽仪进行合成，合成浓度0.25M，根据预设序列计算出各种氨基酸的用量，称取天冬酰胺2.51g溶解在21mL N’N二甲基甲酰胺(DMF)中，称取半胱氨酸1.88g溶解在16mL DMF中，称取谷氨酰胺8.43g溶解在69mL DMF中，称取谷氨酸4.52g溶解在53mL DMF中，称取甘氨酸0.36g溶解在6mL DMF中，称取组氨酸0.58g溶解在6mL DMF中，称取异亮氨酸1.14g溶解在16mL DMF中，称取亮氨酸2.62g溶解在37mL DMF中，称取赖氨酸1.04g溶解在11mL DMF中，称取丝氨酸2.46g溶解在32mL DMF中，称取酪氨酸0.56g溶解在6mL DMF中；在超声中使多肽溶解，溶解后将个试剂管安装至仪器上，另称取14.21g Oxime固体溶解在100mL DMF中，配制成Oxime溶液；另取7.8mL DIC溶液混合在92.2mL DMF中，配置成DIC溶液；其中Oxime和DIC是作为偶联剂使用，最后量取168mL 20％哌啶溶液作为脱保护溶剂；最后称取0.367mg Rink amide AM树脂放置在反应釜内；CEM Liberty Blue全自动多肽仪对于多肽合成的步骤为：溶胀树脂、加入脱保护溶剂、开启微波加热(加热至70℃)、除去脱保护溶剂、清洗3遍、加入对应氨基酸、加入偶联剂、开启微波加热(加热至70℃、反应110秒或者230秒)、清洗三遍为一个氨基酸耦合过程。序列内共31个氨基酸、此过程重复31次后固相合成结束。
切割树脂：在固相合成结束后的树脂内加入10mL裂解液，裂解液内含9.25mL三氟乙酸、0.25mL水、0.25mL 1,2-乙二硫醇、0.25mL三异丙基硅烷；放置在旋转仪上裂解2小时；裂解结束后加入30mL无水***充分混合后、8000rmp离心5分钟，此过程重复三次，后得白色多肽粗产物。切割的位置为树枝上氨基与双苄基的连接处。
粗产物提纯：将粗产物以10mg/mL混合溶剂溶解，混合溶剂内水与乙腈的比例为6:4。随后采用制备液相(大连依利特半制备液相)提纯，流动相A为乙腈、流动相B为水含0.3％TFA，洗脱梯度为水80％-20％、乙腈20％-80％、30分钟，在14分钟左右根据吸收峰接收纯产物。
将得到的纯产物溶液通过旋蒸出去乙腈及大部分水分，保留20mL水，至于液氮内降温，随后放入冻干机内，冷井温度-80℃，真空度1.0Pa，冻干72小时获得纯产物粉末。
进一步，步骤(1)中，配制成11.3mg/mL的多肽水溶液。
进一步，步骤(2)中，配制浓度为15.3mg/mL的4-arm-PEG-Mal水溶液。
进一步，多肽/PEG网络水凝胶溶液浓度为1wt％。
上述在酸性条件下通过网络收缩快速形成水凝胶的方法制得的多肽/PEG网络水凝胶。
本发明具有以下有益效果：
1、本发明填补了从溶液中制备水凝胶的空缺的问题，并且所用材料均为生物相容性极好的材料，其中多肽(C-Switcch-C多肽)自身为线性多肽，在酸性的作用下可以产生α螺旋结构，使水凝胶溶液内网络迅速收缩，形成水凝胶。
2、本发明通过制备在酸性溶液中通过网络收缩的水凝胶，为生物活性细胞及生物相容性良好的水凝胶支架，其具备支撑细胞3D生长的条件。其次通过水凝胶溶液内部网络收缩的作用，可以过滤或者提纯溶液内的活性酶等其他物质，为酶回收问题提供一种方向。
附图说明
图1为C-Switcch-C多肽质谱结果；
图2为C-Switcch-C/PEG网络水凝胶溶液示意图；
图3为C-Switcch-C/PEG网络水凝胶示意图。
具体实施方式
以下对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例1
一种在酸性条件下通过网络收缩快速形成水凝胶的方法，包括以下步骤：
(1)合成在酸性条件下具有α螺旋结构的二聚体多肽(C-Switcch-C多肽)，然后配制成11.3mg/mL的C-Switcch-C多肽水溶液；C-Switcch-C多肽质谱结果如图1所示；C-Switcch-C多肽的氨基酸序列如SEQ ID NO.1所示，序列为：CENQSLEQENSQLKQEISQLEQEIQQLHYGC；
(2)采用4-arm-PEG-Mal配制浓度为15.3mg/mL的4-arm-PEG-Mal水溶液；
(3)分别量取步骤(1)所得C-Switcch-C多肽水溶液和步骤(2)所得4-arm-PEG-Mal水溶液1.5mL，调节pH值至7.5，然后定容至2mL，再将4-arm-PEG-Mal水溶液滴加至C-Switcch-C多肽水溶液上部，并持续振荡，随后放置在摇床上振荡0.4-0.6h，得浓度为1wt％的C-Switcch-C/PEG网络水凝胶溶液，如图2所示；
(4)将步骤(3)所得C-Switcch-C/PEG网络水凝胶溶液调节至酸性，水凝胶溶液收缩，得C-Switcch-C/PEG网络水凝胶，如图3所示。
上述C-Switcch-C多肽采用的合成美国CEM Liberty Blue全自动多肽仪进行合成，合成浓度0.25M，根据预设序列计算出各种氨基酸的用量，称取天冬酰胺2.51g溶解在21mL N’N二甲基甲酰胺(DMF)中，称取半胱氨酸1.88g溶解在16mL DMF中，称取谷氨酰胺8.43g溶解在69mL DMF中，称取谷氨酸4.52g溶解在53mL DMF中，称取甘氨酸0.36g溶解在6mL DMF中，称取组氨酸0.58g溶解在6mL DMF中，称取异亮氨酸1.14g溶解在16mL DMF中，称取亮氨酸2.62g溶解在37mL DMF中，称取赖氨酸1.04g溶解在11mL DMF中，称取丝氨酸2.46g溶解在32mL DMF中，称取酪氨酸0.56g溶解在6mL DMF中；在超声中使多肽溶解，溶解后将个试剂管安装至仪器上，另称取14.21g Oxime固体溶解在100mL DMF中，配制成Oxime溶液；另取7.8mL DIC溶液混合在92.2mL DMF中，配置成DIC溶液；其中Oxime和DIC是作为偶联剂使用，最后量取168mL 20％哌啶溶液作为脱保护溶剂；最后称取0.367mg Rink amide AM树脂放置在反应釜内；CEM Liberty Blue全自动多肽仪对于多肽合成的步骤为：溶胀树脂、加入脱保护溶剂、开启微波加热(加热至70℃)、除去脱保护溶剂、清洗3遍、加入对应氨基酸、加入偶联剂、开启微波加热(加热至70℃、反应110秒或者230秒)、清洗三遍为一个氨基酸耦合过程。
切割树脂：在固相合成结束后的树脂内加入10mL裂解液，裂解液内含9.25mL三氟乙酸、0.25mL水、0.25mL 1,2-乙二硫醇、0.25mL三异丙基硅烷；放置在旋转仪上裂解2小时；裂解结束后加入30mL无水***充分混合后、8000rmp离心5分钟，此过程重复三次，后得白色多肽粗产物。切割的位置为树枝上氨基与双苄基的连接处。
粗产物提纯：将粗产物以10mg/mL混合溶剂溶解，混合溶剂内水与乙腈的比例为6:4。随后采用制备液相(大连依利特半制备液相)提纯，流动相A为乙腈、流动相B为水含0.3％TFA，洗脱梯度为水80％-20％、乙腈20％-80％、30分钟，在14分钟左右根据吸收峰接收纯产物。
将得到的纯产物溶液通过旋蒸出去乙腈及大部分水分，保留20mL水，至于液氮内降温，随后放入冻干机内，冷井温度-80℃，真空度1.0Pa，冻干72小时获得纯产物粉末。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (5)
1.一种在酸性条件下通过网络收缩快速形成水凝胶的方法，其特征在于，包括以下步骤：
（1）合成在酸性条件下具有α螺旋结构的二聚体多肽，然后配制成11-12 mg/mL的多肽水溶液；在酸性条件下具有α螺旋结构的二聚体多肽为C-Switcch-C多肽；所述C-Switcch-C多肽的氨基酸序列如SEQ ID NO.1所示，序列为：CENQSLEQENSQLKQEISQLEQEIQQLHYGC；
（2）采用4-arm-PEG-Mal配制浓度为15-16 mg/mL的4-arm-PEG-Mal水溶液；
（3）分别量取步骤（1）所多肽水溶液和步骤（2）所得4-arm-PEG-Mal水溶液1.5 mL，调节pH值至7.5，然后定容至2 mL，再将4-arm-PEG-Mal水溶液滴加至多肽水溶液上部，并持续振荡，随后放置在摇床上振荡0.4-0.6h，得多肽/PEG网络水凝胶溶液；
（4）将步骤（3）所得多肽/PEG网络水凝胶溶液调节至酸性，线性多肽结构转变为具有α螺旋结构的二聚体多肽结构，网络收缩，得多肽/PEG网络水凝胶。
2.如权利要求1所述的在酸性条件下通过网络收缩快速形成水凝胶的方法，其特征在于，步骤（1）中，配制成11.3 mg/mL的多肽水溶液。
3.如权利要求1所述的在酸性条件下通过网络收缩快速形成水凝胶的方法，其特征在于，步骤（2）中，配制浓度为15.3 mg/mL的4-arm-PEG-Mal水溶液。
4.如权利要求1所述的在酸性条件下通过网络收缩快速形成水凝胶的方法，其特征在于，多肽/PEG网络水凝胶溶液浓度为1wt%。
5.权利要求1-4任一项所述的在酸性条件下通过网络收缩快速形成水凝胶的方法制得的多肽/PEG网络水凝胶。
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