CN114634548A - 一种混合水凝胶的制备方法及其在封装系统中的应用 - Google Patents
一种混合水凝胶的制备方法及其在封装系统中的应用 Download PDFInfo
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Abstract
本发明以自组装肽基水凝胶为基础，根据应用需求对复合凝胶剂的筛选、配方探索确定与肽分子和所负载活性物质的相互作用，提供了提高混合水凝胶的封装效率的混合凝胶配方、混合水凝胶的制备方法及作为封装系统的应用。步骤如下：首先利用固相合成法合成多肽单体序列，再以纯水溶解，经振荡并超声得到多肽水溶液，经冷冻干燥得到短肽粉末，其氨基酸序列为Ac‑X‑F‑F‑NH2；称取一定量短肽粉末，利用有机溶剂溶解制备高浓度短肽储备液，在将储备液以海藻酸盐(Alg)水溶液稀释混匀，静置待短肽形成自组装纳米纤维结构，再以CaCl2溶液促Alg交联，静置得到水凝胶。本发明获得的混合水凝胶用于食品功能因子的封装递送体系既能增强其应用稳定性，又能保证在人体消化吸收过程中的有效释放。
Description
技术领域
本发明涉及生物材料技术领域，具体涉及一种混合水凝胶的制备方法及其在封装系统中的应用。
背景技术
短肽自组装水凝胶是一种新型的软物质材料，它由短肽通过自组装形成，具有生物活性和生物相容性高，易于设计与合成以及对外部刺激能快速响应等优点，它在生物医药，分析检测，组织工程等领域有很高的应用前景。
肽基水凝胶因具有良好的生物相容性，可适用于药物递送、细胞培养、组织工程及创伤愈合、构建3D打印材料等领域。由于应用的需要，人们对水凝胶的优化不仅在于凝胶剂的改性和修饰，还对不同基质的水凝胶进行了复配。以增强力学强度、改善流变学特性、增加对特殊条件的耐受或对调整其物理条件敏感性等为目的对凝胶剂进行改良，使其获得在相关应用领域更优越的性能。通过对肽自组装形成的网络结构进行改善，可以调整水凝胶的力学特性，其中通过与其他凝胶剂的复合来实现肽基水凝胶的凝胶特性优化是较为直接的方法。
水凝胶通常单独用作亲水性生物活性成分的输送。茶多酚、花青素、虾青素等极性活性成分对人体都有抗氧化、抗炎、抗动脉粥样硬化等生理功能，但同时因为活性官能团的存在，在环境中的不稳定性导致其不利于储存和应用，而封装对其在环境中的稳定性有正面作用。本发明通过对混合凝胶剂的筛选，既可以保证肽基水凝胶的生物相容性良好，又能增强凝胶强度，使其应用在食品工业领域中。
发明内容
要解决的技术问题：本发明的目的是以自组装肽基水凝胶为基础，根据应用需求对复合凝胶剂的筛选、配方探索确定与肽分子和所负载活性物质的相互作用，以提高混合水凝胶的封装效率的混合凝胶配方。获得简单，易于制备，成本较低的混合水凝胶配方，及简单快速，水凝胶力学性能较好的交联方式。使得该混合水凝胶用于食品功能因子的封装递送体系既能增强其应用稳定性，又能保证在人体消化吸收过程中的有效释放。
技术方案：
一种具有自组装行为短肽：短肽的氨基酸序列为Ac-X-F-F-NH2，其中X为丝氨酸或苏氨酸，F为苯丙氨酸，Ac为序列N端保护基乙酸酐，NH2为序列C端保护基氨基；
其制备方法为：利用固相合成法合成多肽单体序列，再以纯水溶解，经振荡并超声得到多肽水溶液，经冷冻干燥得到具有自组装行为短肽粉末。
包含上述具有自组装行为短肽的混合水凝胶。
混合水凝胶得制备方法，包括以下步骤：
(1)取具有自组装行为短肽粉末，利用有机溶剂溶解制备高浓度短肽储备液；
(2)将储备液以海藻酸钠(Alg)水溶液稀释混匀得短肽稀释液，静置待短肽形成自组装纳米纤维结构；
(3)以CaCl2溶液促Alg交联，静置3-5min得到混合水凝胶。
进一步的，所述步骤(2)中：Alg水溶液浓度为0.1-0.3mg/mL。
进一步的，所述步骤(2)中：稀释后的短肽浓度范围为≥5mg/mL。
进一步的，所述步骤(2)中：短肽稀释液静置温度及时间分别为4-25℃，3-10min。
进一步的，所述步骤(2)中：CaCl2溶液的浓度为5-30mg/mL，体积为≥1mL。
上述的混合水凝胶或混合水凝胶的制备方法在生物材料水凝封装系统胶领域应用。
有益效果：
1.从扫描电镜可观察到混合水凝胶的微观结构，能明显看出水凝胶内部呈现纤维形成的空间网络结构，由于Alg附着，形成较为粗壮的纤维。
2.从不同配方水凝胶的流变学结果可以看出储能模量均大于耗能模量，所有测试浓度均形成了凝胶状态，混合配方的水凝胶强度比肽基水凝胶的凝胶强度明显增强。
3.从茶多酚封装试验可知，混合水凝胶可以进行茶多酚的封装，封装率显示一定浓度的水凝胶对茶多酚封装效率有限，例如配方为0.8wt％Ac-T-F-F-NH2+0.1％Alg的水凝胶可以封装茶多酚的质量为21.97mg。
附图说明
图1为0.5wt％Ac-S-F-F-NH2+0.1％Alg混合水凝胶的宏观图及SEM图。
图2为0.5wt％Ac-T-F-F-NH2+0.1％Alg混合水凝胶的宏观图及SEM图。
图3为配方分别为0.5wt％Ac-S-F-F-NH2、0.5wt％Ac-T-F-F-NH2、0.5wt％Ac-S-F-F-NH2+0.1％Alg、0.5wt％Ac-T-F-F-NH2+0.1％Alg的混合水凝胶的储能模量图。
图4为浓度为0.8wt％Ac-T-F-F-NH2+0.1％Alg混合水凝胶封装不同浓度茶多酚溶液的封装率结果。
具体实施方式
以下通过实施例对本发明做进一步说明。
实施例1
利用固相合成法合成氨基酸序列为：Ac-S-F-F-NH2的短肽产品(其中S为丝氨酸，F为苯丙氨酸，Ac为序列N端保护基乙酸酐，NH2为序列C端保护基氨基)，经超声、冻干处理得到产品短肽粉末。称取产品短肽5mg于样品瓶中，加入200μL六氟异丙醇制备为浓度25mg/mL肽储备液，加浓度为0.1％的Alg溶液至体系总重量为1g，得到浓度为0.5wt％Ac-S-F-F-NH2+0.1％Alg的混合溶液，在4℃下静置5min等待短肽分子间自组装以及Alg在肽纤维上的附着。在混合溶液呈现出倒置不流动的状态后，将1mL浓度为20mg/mL CaCl2溶液加入样品瓶，静置10min，可形成混合水凝胶。
由该方法制备的水凝胶通过SEM进行水凝胶的微观形貌的观察。取0.1g肽基水凝胶进行冷冻干燥，取适量置于导电胶上，喷金5min后，通过SEM观察。从图1中能明显看出在CaCl2溶液促进交联后，水凝胶发生一定程度的紧缩，水凝胶内部呈现纤维形成的空间网络结构，并且纤维较肽基水凝胶更为粗壮，表面不光滑。
实施例2
利用固相合成法合成氨基酸序列为：Ac-T-F-F-NH2的短肽产品(其中T为苏氨酸，F为苯丙氨酸，Ac为序列N端保护基乙酸酐，NH2为序列C端保护基氨基)，经超声、冻干处理得到产品短肽粉末。称取产品短肽5mg于样品瓶中，加入50μL六氟异丙醇制备为浓度100mg/mL肽储备液，加浓度为0.1％的Alg溶液至体系总重量为1g，得到浓度为0.5wt％Ac-T-F-F-NH2+0.1％Alg的混合溶液，在4℃下静置5min等待短肽分子间自组装以及Alg在肽纤维上的附着。在混合溶液呈现出倒置不流动的状态后，将1mL浓度为20mg/mL CaCl2溶液加入样品瓶，静置10min，可形成混合水凝胶。
由该方法制备的水凝胶通过SEM进行水凝胶的微观形貌的观察。取0.1g肽基水凝胶进行冷冻干燥，取适量置于导电胶上，喷金5min后，通过SEM观察。通过图2对比丝氨酸合成的肽基混合水凝胶围观形貌，苏氨酸合成的肽基混合水凝胶的纤维长度更长，网络结构更连贯，所以在宏观上可观察到在CaCl2溶液促进交联后，水凝胶发生较为明显的体积紧缩。
实施例3
利用固相合成法合成氨基酸序列为：Ac-T-F-F-NH2(其中T为苏氨酸，F为苯丙氨酸，Ac为序列N端保护基乙酸酐，NH2为序列C端保护基氨基)的短肽产品。将粗肽经过超声、振荡处理后冷冻干燥得到产品短肽粉末。分别称取短肽产品40mg于样品瓶中，共8份，加入适量六氟异丙醇制备浓度为100mg/mL的肽储备液。称取茶多酚固体溶解在一定体积0.1％Alg溶液中，再将混合溶液加入肽储备液，制备得到浓度分别为0、2、5、10、17.5、25、35、50mg/mL茶多酚-Alg混合溶液。将不同浓度的茶多酚-Alg混合溶液加入肽储备液中至体系总质量为5g，在4℃下静置1H，至倒置样品瓶不流动。加入适量的20mg/mL CaCl2溶液促样品中Alg交联，静置5min，即形成封装不同浓度茶多酚的混合水凝胶，其配方为0.8wt％Ac-T-F-F-NH2+0.1％Alg。
对封装不同浓度茶多酚溶液的混合水凝胶进行封装率测定。将水凝胶样品整块取出，使用纯水将其淋洗3-5次至淋洗液透明，淋洗过程不可破坏凝胶结构。将淋洗过的凝胶经冷冻干燥得到固体，称取凝胶固体10mg于试管中，加纯水至5ml。在功率200W条件下超声分散2min，冰浴保持超声过程温度不高于40℃。在12000rpm条件下离心20min，取适量上层清液于试管中，依据国标GBT8313-2018的方法测定茶多酚含量，以此结果判断肽基水凝胶的封装率。可以发现低浓度的茶多酚封装效率较高，茶多酚溶液浓度超过25mg/mL后，封装率下降，说明该肽基水凝胶产品可以有效封装一定量的茶多酚，高浓度茶多酚溶液中的茶多酚无法被封装。
对比例1
利用固相合成法合成氨基酸序列为：Ac-S-F-F-NH2(其中S为丝氨酸，F为苯丙氨酸，Ac为序列N端保护基乙酸酐，NH2为序列C端保护基氨基)的短肽产品。经超声、冻干处理得到产品短肽粉末。称取短肽5mg于样品瓶中，加入200μL六氟异丙醇制备为浓度25mg/mL肽储备液，在样品中添加纯水至体系总重量为1g，得到配方浓度为0.5wt％的Ac-S-F-F-NH2溶液，在4℃下静置5min，待短肽分子间自组装形成肽纳米纤维，并进一步组装交联形成空间网络结构。在样品呈现出倒置不流动的状态后，即形成肽基水凝胶。
对比例2
利用固相合成法合成氨基酸序列为：Ac-T-F-F-NH2(其中T为苏氨酸，F为苯丙氨酸，Ac为序列N端保护基乙酸酐，NH2为序列C端保护基氨基)的短肽产品。经超声、冻干处理得到产品短肽粉末。称取短肽5mg于样品瓶中，加入200μL六氟异丙醇制备为浓度25mg/mL肽储备液，在样品中添加纯水至体系总重量为1g，得到配方浓度为0.5wt％的Ac-T-F-F-NH2溶液，在4℃下静置5min，待短肽分子间自组装形成肽纳米纤维，并进一步组装交联形成空间网络结构。在样品呈现出倒置不流动的状态后，即形成肽基水凝胶。
将实施例1、2中的混合水凝胶及上述两种肽基水凝胶进行振幅扫描及频率扫描，混合水凝胶产品的线性黏弹区范围比肽基水凝胶产品大，在振幅为0.5％的小形变，频率为0.1-10Hz的条件下，测定不同配方水凝胶的储能及耗能模量。通过与实施例1、2的产品对比，从图3可以看出混合水凝胶的凝胶强度比肽基水凝胶明显增强。这可能是由于Alg的附着，加固了肽基纤维。其中短肽分子为Ac-S-F-F-NH2的凝胶强度较弱，所以在该频率范围内出现模量的明显变化，结构易被破坏，证明其力学性能较差，但由Alg参与的混合凝胶其模量在常用频率下稳定显示出较好的凝胶强度。
Claims (8)
1.一种具有自组装行为短肽，其特征在于，短肽的氨基酸序列为Ac-X-F-F-NH2，其中X为丝氨酸或苏氨酸，F为苯丙氨酸，Ac为序列N端保护基乙酸酐，NH2为序列C端保护基氨基；
其制备方法为：利用固相合成法合成多肽单体序列，再以纯水溶解，经振荡并超声得到多肽水溶液，经冷冻干燥得到具有自组装行为短肽粉末。
2.包含权利要求1所述具有自组装行为短肽的混合水凝胶。
3.根据权利要求2所述的混合水凝胶，其特征在于，制备方法包括以下步骤：
(1)取具有自组装行为短肽粉末，利用有机溶剂溶解制备高浓度短肽储备液；
(2)将储备液以海藻酸盐(Alg)水溶液稀释混匀得短肽稀释液，静置待短肽形成自组装纳米纤维结构；
(3)以CaCl2溶液促Alg交联，静置3-5min得到混合水凝胶。
4.根据权利要求3所述的一种混合水凝胶的制备方法，其特征在于：所述步骤(2)中Alg水溶液浓度为0.1-0.3mg/mL。
5.根据权利要求3所述的一种混合水凝胶的制备方法，其特征在于：所述步骤(2)中稀释后的短肽浓度范围为≥5mg/mL。
6.根据权利要求3所述的一种混合水凝胶的制备方法，其特征在于：所述步骤(2)中短肽稀释液静置温度及时间分别为4-25℃，3-10min。
7.根据权利要求3所述的一种混合水凝胶的制备方法，其特征在于：所述步骤(2)中CaCl2溶液的浓度为5-30mg/mL，体积为≥1mL。
8.根据权利要求2所述的混合水凝胶或者权利要求3-7任一项所述的混合水凝胶的制备方法所制备的混合水凝胶在封装系统领域应用。
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