CN113956326A - 一种短肽单体,结构自愈型肽基水凝胶及其应用 - Google Patents
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Abstract
本发明提供了一种短肽单体,结构自愈型肽基水凝胶及其应用。所述的肽基水凝胶是通过合成的两端封端的三肽,其序列如下:Ac‑T‑F‑F‑NH2,在溶液中形成自组装体,在一定条件下肽的自组装小体继续组装形成纳米纤维形成空间三维网络结构。本发明提供的水凝胶具有较好的结构恢复能力和被生物降解能力,其制备方法所用原料均具有良好的生物相容性,在水凝胶封装过程以及人体摄入过程都有合适的流变特性便于应用。
Description
技术领域
本发明涉及生物材料技术领域,具体涉及一种短肽单体,结构自愈型肽基水凝胶及其应用。
背景技术
由于肽基水凝胶具有高持水能力、多微孔结构、可调的机械性能、良好的生物相容性等性能,以及肽基凝胶剂通常具有主链可整合(调整枝节程度)、侧链可修饰的优势,可调整适用于不同领域。特别是,肽基水凝胶又可以设计得到肽结合位点的结构,所以在药物分子递送、组织工程以及创伤愈合方面具有极大的应用研究价值。短肽在促组装驱动力,如静电力、疏水力、氢键、π-π堆叠等相互作用下封包水相形成肽基水凝胶。
在肽基凝胶剂的设计上可以根据对pH敏感、对温度敏感等要求设计肽序,利用由氨基酸侧链电负性差异或极性不同的氨基酸侧链设计合成配对肽或两亲性多肽。但是目前的研究多集中于长肽链多肽或多聚肽,对于具有更好生物相容性的短肽水凝胶研究较少。仅有的研究成果显示成胶方式较为复杂,包括有自组装模板、pH调节或金属离子驱动等方式。在应用中这些条件可能会对结果产生明显影响,故本发明是具有更简单的成胶方式,且对应用过程影响较小的新产品的开发。
发明内容
要解决的技术问题:本发明的目的在于针对物质递送,设计发明一种具有结构恢复性的多肽单体,在一定条件下形成肽基水凝胶,利用其良好的生物相容性和结构可自愈的性能,开发其作为药物分子或食品功能因子的递送体系。该肽基水凝胶的肽序长度短,易于制备,成本较低,成交方式简单快速,可用于极性或非极性物质的封装递送体系。
技术方案:一种具有自组装行为的短肽单体,所述短肽单体的氨基酸序列通式为苏氨酸-苯丙氨酸-苯丙氨酸(TFF)。
优选的,所述的短肽单体的N端进行了乙酰化,C端进行氨基化。
优选的,所述短肽单体通过以下方法制备而成: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单体水溶液,经冷冻干燥得到短肽单体粉末。
上述的短肽单体在制备肽基水凝胶中的应用。
优选的,所述超声功率为50-80W,超声温度低于40℃,超声时间为30min。
一种肽基水凝胶,所述的水凝胶是将权利要求1所述的多肽单体加入有机溶剂制备高浓度短肽储备液,在将储备液以纯水稀释混匀得到短肽水溶液,静置形成水凝胶;其中,所述短肽单体通过苯丙氨酸侧链苯环间产生的π-π堆积作用而形成有序的短肽单体反向平行排列,进而自组装为纤维链状结构,所组装的纤维结构的直径在30-50nm之间。
优选的,所述有机溶剂为六氟异丙醇、异丙醇或丙酮中的任意一种或两种及两种以上的组合物。
优选的,所述短肽储备液的浓度范围为100-25mg/mL,所述短肽水溶液浓度范围为大于0.5wt%。
优选的,所述静置温度及时间分别为4-25℃,5-120min。
上述的肽基水凝胶在制备可注射手术填充物、生物纳米材料、药物递送、组织修复和细胞支架中的应用。
有益效果:本发明的具有以下优点
1.本发明的短肽,该短肽具有一端亲水、一端疏水的两亲性特征,该短肽在生理条件下自组装形成纳米纤维结构且形成水凝胶,丰富了水凝胶形成的种类;
2.本发明的肽基水凝胶是由三肽TFF组装而成,操作简捷,快速,制备过程中无需引入任何引发剂和交联剂,安全无毒且环保;
3.本发明的肽基水凝胶,其可由肽分子间的自组装驱动形成水凝胶,其肽序列简单,肽链长度短,得到的水凝胶生物相容性好且具有结构破坏后可自愈的特性;
4.本发明的水凝胶其对极性和非极性小分子均具有封装性能,有助于肽基水凝胶材料在多个领域的开发利用。
附图说明:
图1为肽基凝胶剂,即三肽Ac-T-F-F-NH2的质谱图;
图2为浓度为0.5wt%的肽溶液形成水凝胶的宏观图及TEM、SEM图;
图3为浓度分别为0.3、0.5、0.8、1.0、1.5wt%的肽基水凝胶的储能模量图,及上述浓度形成水凝胶的宏观图;
图4为浓度为0.5wt%的肽基水凝胶结构破坏恢复试验宏观图及流变学三段式结构破坏恢复模量图。
具体实施方式
实施例1
利用固相合成法合成氨基酸序列为:Ac-T-F-F-NH2的多肽单体,将吹干乙醚的粗多肽单体中加入适量的纯水,在50W功率下,超声处理30min,过程中添加冰块保证超声温度不高于40℃。然后在气浴振荡器中振荡过夜,使肽完全溶解后进行冷冻干燥得到产品短肽粉末。
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,加纯水至体系总重量为1g,得到浓度为0.5wt%的肽溶液,在4℃下静置10min。通过倒置样品瓶的方式判断溶液是否形成肽基水凝胶,若样品瓶倒置样品不滑落,即形成水凝胶。由该方法制备的水凝胶通过TEM、SEM进行水凝胶的微观形貌的观察。取肽基水凝胶100μL,用纯水稀释至200μL,通过涡旋使肽基水凝胶分散在纯水中,吸取20μL分散相滴于碳膜覆盖的铜网上,静置干燥,通过TEM观察。取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。从结果能明显看出水凝胶内部呈现纤维形成的空间网络结构,并且纤维直径在30-50nm之间。
实施例2
利用固相合成法合成氨基酸序列为:Ac-T-F-F-NH2的多肽单体,将吹干乙醚的粗多肽单体中加入适量的纯水,在50W功率下,超声处理30min,过程中添加冰块保证超声温度不高于40℃。然后在气浴振荡器中振荡过夜,使肽完全溶解后进行冷冻干燥得到产品短肽粉末。分别称取产品短肽3、5、8、10、1.5mg于样品瓶中,加入150μL六氟异丙醇制备为不同浓度的肽储备液,加纯水至体系总重量为1g,得到浓度分别为0.3、0.5、0.8、1.0、1.5wt%的肽溶液,在4℃下静置1H。通过倒置样品瓶的方式判断溶液是否形成肽基水凝胶,若样品瓶倒置样品不滑落,即形成水凝胶。
对不同浓度的肽基水凝胶进行振幅扫描及频率扫描,发现产品的线性黏弹区随产品浓度升高而增大,在振幅为0.5%的小形变,频率为0.1-10Hz的条件下,测定不同浓度水凝胶的储能及耗能模量。从结果可以看出储能模量均大于耗能模量,故所有测试浓度均形成了凝胶状态,并且随浓度的增大,凝胶强度增大。其中由于0.3wt%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,这是结构被破坏的证明,与宏观观察一致。
实施例3
利用固相合成法合成氨基酸序列为:Ac-T-F-F-NH2的多肽单体,将吹干乙醚的粗多肽单体中加入适量的纯水,在50W功率下,超声处理30min,过程中添加冰块保证超声温度不高于40℃。然后在气浴振荡器中振荡过夜,使肽完全溶解后进行冷冻干燥得到产品短肽粉末。称取产品短肽5mg于样品瓶中,加入50μL六氟异丙醇制备浓度为100mg/mL的肽储备液。加纯水至体系总重量为1g,得到浓度为0.5wt%的肽溶液,在4℃下静置10min。通过倒置样品瓶的方式判断溶液是否形成肽基水凝胶,若样品瓶倒置样品不滑落,即形成水凝胶。
将该水凝胶样品进行流变学结构破坏恢复试验。第一阶段,将肽基水凝胶置于振幅为0.01%的极小形变和频率为1Hz的条件下稳定结构180s;第二阶段,使振幅增至50%大形变量和频率为1Hz的条件下,60s时间内破坏原有结构;第三阶段,使振幅恢复至0.01%的极小形变和频率为1Hz的条件测定模量变化,判断结构恢复情况,破坏恢复过程重复一次,过程时长为1500s。得到在经历两次破坏后,1500s时结构可以恢复至样品未破坏时的49.7%,具有一定的结构破坏后自愈能力,与宏观观察到的结果一致。
Claims (10)
1.一种具有自组装行为的短肽单体,其特征在于,所述短肽单体的氨基酸序列通式为苏氨酸-苯丙氨酸-苯丙氨酸。
2.根据权利要求1所述的多肽,其特征在于,所述的短肽单体的N端进行了乙酰化,C端进行氨基化。
3.根据权利要求1所述的短肽单体,其特征在于,所述短肽单体通过以下方法制备而成: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单体水溶液,经冷冻干燥得到短肽单体粉末。
4.权利要求1所述的短肽单体在制备肽基水凝胶中的应用。
5.根据权利要求3中所述的短肽单体,其特征在于:所述超声功率为50-80W,超声温度低于40℃,超声时间为30min。
6.一种肽基水凝胶,其特征在于,所述的水凝胶是将权利要求1所述的多肽单体加入有机溶剂制备高浓度短肽储备液,在将储备液以纯水稀释混匀得到短肽水溶液,静置形成水凝胶;其中,所述短肽单体通过苯丙氨酸侧链苯环间产生的π-π堆积作用而形成有序的短肽单体反向平行排列,进而自组装为纤维链状结构,所组装的纤维结构的直径在30-50nm之间。
7.根据权利要求6中所述肽基水凝胶,其特征在于:所述有机溶剂为六氟异丙醇、异丙醇或丙酮中的任意一种或两种及两种以上的组合物。
8.根据权利要求6中所述肽基水凝胶,其特征在于:所述短肽储备液的浓度范围为100-25mg/mL,所述短肽水溶液浓度范围为大于0.5wt%。
9.根据权利要求6中所述肽基水凝胶,其特征在于:所述静置温度及时间分别为4-25℃,5-120min。
10.权利要求6-9任一项所述的肽基水凝胶在制备可注射手术填充物、生物纳米材料、药物递送、组织修复和细胞支架中的应用。
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