CN113462640B - 一种用于调控细胞铺展速率的笼闭配体的制备方法 - Google Patents
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Abstract
本发明公开了一种用于调控细胞铺展速率的笼闭配体的制备方法，包括：将配体分子加入DMF溶剂中，充分搅拌溶解制得配体分子溶液，备用；将缩合剂和缩合活化剂加入DMF溶剂中，充分搅拌溶解；再加入小分子胺，充分搅拌溶解得到混合溶液，备用；将等体积的配体分子溶液和混合溶液混合，搅拌，在下震荡反应12‑18h；采用双蒸水稀释上述溶液到适宜浓度，并接枝到水凝胶中或涂层表面；去除水凝胶中或涂层表面的残留的交联剂、交联活化剂及副产物，得到可酶解的笼闭配体。所述配体分子为含有RGD三肽片段或结合细胞粘附的整合素与钙粘蛋白多肽或模拟多肽的化合物或含有巯基、叠氮、氨基、羧基其中之一的活性基团的天然或合成化合物。
Description
技术领域
本发明涉及生物材料技术领域，尤其是一种用于调控细胞铺展速率的笼闭配体的制备方法。
背景技术
众所周知，细胞通过多种粘附蛋白与细胞外基质粘附。1984年，科学家从纤连蛋白中简化出整合素受体可识别的最简三肽Arg-Gly-Asp(RGD)，从此，RGD成为让材料提高细胞亲和度的优秀策略，被称为“细胞胶水”。RGD与整合素的结合是通过精氨酸侧链的胍基与α亚基结合，天冬氨酸侧链的羧基与β亚基结合，如果将这两个官能团之一抑制，将会使结合失效。经上述研究，通过这一特性可设计RGD活性笼闭的材料，通过对RGD活性的调控可调控细胞的粘附、迁移、分化等行为。另外，通过这一策略可进行细胞力学信号转导的研究，并且可指导超高生物相容性材料的设计策略。
目前，现有技术中的RGD活性智能响应材料的应激源主要包括：电信号、光信号、磁场、少量有酶促响应的研究。其中，电信号和光信号对细胞本身有不可预估的影响，磁响应材料需要磁性纳米颗粒。这一领域的发展趋向于对细胞本身无影响的信号，即生物相容性的提高。
因此，急需要提出一种降解RGD的抑制基团，通过对抑制基团的活性调节可实现细胞的降解快慢差异，从而实现细胞粘附的差异；通过该抑制基团实现或快或慢的粘附迁移速率，以及后续成骨分化、成脂分化的区别。
发明内容
针对上述问题，本发明的目的在于提供一种用于调控细胞铺展速率的笼闭配体的制备方法，本发明采用的技术方案如下：
一种用于调控细胞铺展速率的笼闭配体的制备方法，包括以下步骤：
将配体分子加入DMF溶剂中，充分搅拌溶解制得配体分子溶液，备用；所述配体分子为含有RGD三肽片段或结合细胞粘附的整合素与钙粘蛋白多肽或模拟多肽的化合物或含有巯基、叠氮、氨基、羧基其中之一的活性基团的天然或合成化合物；
将1-10当量的缩合剂和缩合活化剂充分溶解在DMF溶剂中，充分搅拌溶解；再加入1-10当量的小分子胺，充分搅拌溶解得到混合溶液，备用；
将等体积的配体分子溶液和混合溶液混合，搅拌，在室温下震荡反应12-18h；
采用双蒸水稀释上述溶液，并使其浓度在0.001mmol/L-1mol/L，并接枝到水凝胶中或涂层表面；
去除水凝胶中或涂层表面的残留的交联剂、交联活化剂及副产物，得到可酶解的笼闭配体。
优选地，所述缩合剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐。
优选地，所述缩合活化剂为4-二甲氨基吡啶或/和1-羟基苯并三唑。
进一步地，所述小分子胺为3,3,3-三氟丙胺、2,2,3,3,3-五氟丙胺、特戊胺、叔丁胺、环丙胺、2,4-二硝基苯胺、苯胺、N,N-二甲基磺酰胺、4-三氟甲基苯胺、4-硝基苯胺其中之一或其中几个。
进一步地，所述配体分子溶液的浓度为2.5mM；所述含有RGD三肽片段的天然或合成化合物、小分子胺、缩合剂，缩合活化剂摩尔比为1:10:10:10。
优选地，所述水凝胶或涂层采用聚乙二醇、聚缩水甘油、聚乙烯醇、葡聚糖、海藻酸其中之一的材料。
与现有技术相比，本发明具有以下有益效果：
(1)本发明巧妙地采用了含有RGD三肽片段或结合细胞粘附的整合素与钙粘蛋白多肽或模拟多肽的化合物或含有巯基、叠氮、氨基、羧基其中之一的活性基团的天然或合成化合物作为配体分子，其利用配体基团的保护技术，制备具有可控酶响应能力的笼闭多肽配体，用于修饰界面生物材料，调控细胞粘附与骨架构建速率，可用于组织工程中细胞铺展、迁移、增殖、分化等行为的调控；
(2)本发明采用RGD等配体分子可与细胞粘附受体(整合素与钙粘蛋白等)相互作用，即可对多数细胞产生粘附作用。其中，RGD与小分子胺反应的过程可控，所选缩合剂与缩合活化剂以及产生的副产物脲可溶于水，在清洗过程中可被除去。
(3)本发明的多肽配体设计官能基团便利，可以方便地将笼闭的配体修饰在水凝胶或涂层基底材料上。活性笼闭部分酰胺键活性的调节可产生细胞耐受性的差异，细胞外分泌的酶对酰胺键的水解速率的差异引起细胞在水凝胶表面粘附速度的差异。
(4)本发明避免了外加刺激条件如光、电场等对细胞产生潜在影响的因素。
(5)本发明所采用调控细胞粘附与铺展速率的笼闭配体，生物相容性好，化学结构稳定，制备方法简单，条件温和，可广泛用于各类生物材料的改性；
综上所述，本发明具有工艺简单、稳定可靠等优点，在生物材料技术领域具有很高的实用价值和推广价值。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需使用的附图作简单介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对保护范围的限定，对于本领域技术人员来说，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本发明中HUVEC、MSC、L929细胞在水凝胶上培养18小时后细胞铺展面积分布统计图。
图2为本发明中RGD cage的结构示意图。
图3为本发明中L929细胞在四种PEG-RGD cage水凝胶上培养18小时后细胞铺展示意图。
图4为本发明中L929细胞在四种PEG-RGD cage水凝胶上培养36小时后细胞铺展示意图。
图5为本发明中L929细胞在水凝胶上培养细胞面积趋势图。
具体实施方式
为使本申请的目的、技术方案和优点更为清楚，下面结合附图和实施例对本发明作进一步说明，本发明的实施方式包括但不限于下列实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本申请保护的范围。
实施例
如图1至图5所示，本实施例提供了一种用于调控细胞铺展速率的笼闭配体的制备方法，包括以下步骤
第一步，将配体分子加入DMF溶剂中，充分搅拌溶解制得配体分子溶液，备用；所述配体分子为含有RGD三肽片段或结合细胞粘附的整合素与钙粘蛋白多肽或模拟多肽的化合物或含有巯基、叠氮、氨基、羧基其中之一的活性基团的天然或合成化合物。
第二步，将1-10当量的缩合剂和缩合活化剂充分溶解在DMF溶剂中，充分搅拌溶解；再加入1-10当量的小分子胺，充分搅拌溶解得到混合溶液，备用。在此，缩合剂选用EDCI(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)。缩合活化剂选用DMAP(4-二甲氨基吡啶或/和1-羟基苯并三唑)。另外在本实施例中，小分子胺选用但不限于3,3,3-三氟丙胺，2,2,3,3,3-五氟丙胺，特戊胺，叔丁胺，环丙胺，2,4-二硝基苯胺，苯胺，N,N-二甲基磺酰胺，4-三氟甲基苯胺，4-硝基苯胺或其组合。
第三步，将等体积的配体分子溶液和混合溶液混合，搅拌，在室温下震荡反应12-18h；配体分子溶液的浓度为2.5mM；所述含有RGD三肽片段的天然或合成化合物、小分子胺、缩合剂，缩合活化剂摩尔比为1:10:10:10。
第四步，采用双蒸水稀释上述溶液，并使其浓度至0.001mmol/L-1mol/L，并接枝到水凝胶中或涂层表面。在本步骤中，笼闭配体修饰的水凝胶或涂层材料为各种生物材料，包括但不限于聚乙二醇、聚缩水甘油、聚乙烯醇、葡聚糖、海藻酸。
第五步，去除水凝胶中或涂层表面的残留的交联剂、交联活化剂及副产物，得到笼闭配体。
下面简要说明其应用过程：
步骤S1：配置PEG(聚乙二醇)水溶液500mg/mL，光引发剂：70％乙醇溶液20mg/mL。取上述步骤三所述RGD反应溶液稀释至1mM。
步骤S2：将PEG水溶液、光引发剂、双蒸水、RGD按体积比80:9:1:10混合均匀，在紫外灯下照射15分钟成胶制得笼闭RGD修饰的PEG水凝胶。Cage I为3,3,3-三氟丙胺，Cage II为2,2,3,3,3-五氟丙胺，Cage III为特戊胺，Cage IV为环丙胺。
测试1：
取实施例中cage I的水凝胶清洗，分别接种HUVEC、MSC和L929细胞，培养18小时后显微镜拍照并统计细胞面积。从图中可以看出：细胞接种4小时后，在不同的可酶解的笼闭配体修饰的PEG凝胶表面。其测试结果如图1所示，说明三种细胞在cage I笼闭配体修饰的RGD-PEG凝胶表面接种18小时后均表现出对笼闭配体的降解能力，且细胞铺展速率慢于未被笼闭配体修饰的RGD-PEG凝胶，L929表现出对cage I笼闭配体相对最快的降解速率。
测试2：
取实施例中的水凝胶清洗，分别接种L929细胞，培养18小时后拍照。如图3所示，其说明Cage I、Cage II、Cage III、Cage IV笼闭配体修饰的RGD-PEG凝胶对L929细胞的黏附铺展均体现出一定抑制能力。
测试3：
取实施例中的水凝胶清洗，分别接种L929细胞，培养36小时后拍照。如图4所示，其说明L929细胞对Cage I、Cage II、Cage III、Cage IV笼闭配体均体现出一定的降解能力，并且对Cage I、Cage II降解速率要快于Cage III、Cage IV。
测试4：
取实施例中的水凝胶清洗，分别接种L929细胞，统计36小时内细胞铺展面积。如图5所示，其说明了L929细胞的黏附铺展受笼闭配体结构的调控，且对缺电性结构笼闭配体的降解速率快于富电性缺电性结构笼闭配体。因此，笼闭配体可以通过细胞酶解笼闭基团的速率，调节细胞粘附铺展速率。
上述实施例仅为本发明的优选实施例，并非对本发明保护范围的限制，但凡采用本发明的设计原理，以及在此基础上进行非创造性劳动而作出的变化，均应属于本发明的保护范围之内。
Claims (1)
1.一种用于调控细胞铺展速率的笼闭配体的制备方法，其特征在于，包括以下步骤：
将配体分子加入DMF溶剂中，充分搅拌溶解制得配体分子溶液，备用；所述配体分子含有RGD三肽片段；
将1-10当量的缩合剂和缩合活化剂充分溶解在DMF溶剂中，充分搅拌溶解；再加入1-10当量的小分子胺，充分搅拌溶解得到混合溶液，备用；
将等体积的配体分子溶液和混合溶液混合，搅拌，在室温下震荡反应12-18h，采用双蒸水稀释上述溶液，得到浓度为0.001 mmol/L - 1 mol/L的稀释溶液，并接枝到水凝胶中；
去除水凝胶中残留的交联剂、交联活化剂及副产物，得到可酶解的笼闭配体；所述配体分子溶液的浓度为2.5 mM；所述RGD三肽片段、小分子胺、缩合剂，缩合活化剂摩尔比为1:10:10:10；所述缩合剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，所述缩合活化剂为4-二甲氨基吡啶或/和1-羟基苯并三唑；所述小分子胺为3,3,3-三氟丙胺、2,2,3,3,3-五氟丙胺、特戊胺、环丙胺其中之一；
使用时将PEG水溶液、光引发剂、双蒸水、RGD按体积比80:9:1:10混合均匀，在紫外灯下照射15分钟成胶制得笼闭RGD修饰的PEG水凝胶；所述PEG水溶液 500 mg/mL，所述光引发剂为70%乙醇溶液20 mg/mL。
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