CN106397545B - 一种水凝胶材料及其制备方法和应用 - Google Patents
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Abstract
本发明属于材料制备领域，具体提供了一种短肽，所述短肽序列为：（精氨酸‑丙氨酸‑天冬氨酸‑丙氨酸）4‑异亮氨酸‑赖氨酸‑异亮氨酸‑赖氨酸‑异亮氨酸‑赖氨酸‑缬氨酸‑丙氨酸‑缬氨酸。本发明同时提供了将短肽配置成短肽水溶液自组装形成所述的水凝胶材料，该材料是在RADA16‑I序列的基础上，连接多个酸性和碱性氨基酸，通过非共价键作用结合形成。本发明制备的水凝胶材料有促进神经细胞轴突伸展和神经再生的功能，材料生物相容性好，可实现原位负载细胞/活性分子以及体内原位注射，对神经再生及修复具有显著效果，在包括神经细胞在内的细胞三维培养、组织工程生物支架材料和药物载体领域，具有广阔的应用前景和临床价值。
Description
技术领域
本发明属于材料制备领域，更具体地，涉及一种水凝胶材料及其制备方法和应用。
背景技术
三维多孔纳米支架能够模仿天然细胞外基质结构，为细胞生长提供支持，广泛应用于生物医学、组织工程学等研究领域。几乎所有的组织细胞在体内都是在三维条件下生长，细胞被包裹在由胶原纤维作为主要成分组成的纳米纤维水凝胶之中，此外，细胞外基质中还含有大量的不溶性基质蛋白和可溶性生长因子。但目前研究中细胞培养技术中所采用的大多都是二维培养，即细胞在培养板或者培养皿上进行培养，这与体内生长环境相差较大，影响细胞生长，甚至会引起细胞基因或者功能变化。如何体外构建具有模仿天然细胞外基质结构和功能的人工支架材料，为细胞提供三维生长环境一直是生物、医学以及材料工程领域基础研究和产品开发的热点。
自组装短肽RADA16-I是4个重复的精氨酸-丙氨酸-天冬氨酸-丙氨酸序列，即（精氨酸-丙氨酸-天冬氨酸-丙氨酸）4，当其水溶液调节至pH=7时会形成由纳米纤维网络构成的水凝胶，与天然细胞外基质结构很相似，作为组织工程支架、药物载体和止血材料在生物医学工程领域有广泛的应用，是自组装短肽水凝胶材料的典型代表。但是一个显著的缺点是该材料水溶液具有明显的酸性(pH=3～4)，不能与细胞悬液和活性分子直接混合原位成凝胶，即很难实现细胞包埋在RADA 16-I水凝胶中进行三维生长。
目前关于细胞培养的报道仍是采用先制备水凝胶然后在其表面种植细胞的二维培养模式，不能实现真正的三维细胞培养。而将该材料直接注射体内修复损伤和用于止血时较低的pH会对宿主组织造成损害。自1993年首次报道该材料以来，人们对RADA16-I进行了大量的研究，但是对于其酸性的缺点仍不能有效解决。
发明内容
本发明根据目前自组装水凝胶材料中的不足，提供了一种水凝胶材料。
本发明的另一目的在于提供上述水凝胶材料的制备方法。
本发明的另一目的在于提供上述水凝胶材料的应用。
本发明的技术目的通过以下技术方案实现：
本发明提供了一种短肽，所述短肽序列为：（精氨酸-丙氨酸-天冬氨酸-丙氨酸）4-异亮氨酸-赖氨酸-异亮氨酸-赖氨酸-异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸。
本发明同时保护包含所述短肽的水凝胶材料。
本发明所提供的水凝胶材料，是短肽先与碱液混合，再与缓冲液混合后，在中性pH条件下自组装形成，该短肽是在RADA16-I 序列的基础上，连接多个酸性和碱性氨基酸，然后通过非共价键作用结合形成具有特定功能的多肽水凝胶材料。
本发明是以低分子量自组装短肽为基础的水凝胶，为细胞三维生长提供空间，用于构建组织工程支架。本材料可在常温中性pH条件下形成纳米纤维水凝胶，模仿了天然细胞外基质结构，具有良好的理化性能和生物相容性，支持细胞三维生长，满足组织工程支架的要求，而且可负载活性分子药物，例如生长因子、短肽药物。可广泛应用于软骨、血管、神经、皮肤等人工器官的再生和损伤的修复。
本发明同时提供所述的水凝胶材料的方法，包括将短肽配置成短肽水溶液，先与碱液混合，调节pH得到pH中性溶液，然后再与缓冲液混合，自组装形成所述水凝胶材料。
所述碱液为NaOH溶液或Trsi-base溶液；所述缓冲液为PBS溶液或DMEM溶液。
优选地，所述短肽水溶液的浓度为5~15 mg/mL。
优选地，所述碱液的浓度为0.1~1 mol/mL，所述pH为7~7.4。
优选地，所述短肽水溶液与缓冲液混合体积比为（1~5）：1。
更优选地，所述短肽水溶液与缓冲液混合体积比为2：1。
本发明短肽序列的合成是采用了现有的接枝方向获得，由于目前未有成型的理论支持何种改性（即接枝序列和接枝位置）能够获得真正在中性条件下合成的自组装多肽水凝胶，不同的序列排列方式不同，电荷分布也不同，同时结构内部自组装方式均会影响最终的接枝效果，因此，上述接枝物的具体接枝位点和接枝序列排布，对最终效果影响非常大。
与现有多肽自组装水凝胶相比，本发明在生理条件下（pH为7-7.4）即可形成三维多孔水凝胶，由直径约20 nm的纳米纤维网络构成，凝胶网络结构稳定，可为细胞三维培养提供支撑作用。将含有功能性氨基酸序列引入，能够促进细胞生长、粘附，同时也可负载活性分子药物，如生长因子、短肽药物，获得控制释放。也可注射使用，操作简便，是非常理想的生物材料。
本发明所述的中性pH下自组装成的水凝胶材料，且使用过程都是中性的，不会对细胞核宿主组织产生危害，能够用于真正的三维培养细胞，方法为将细胞悬液与所述短肽材料进行混合，得到包埋细胞的水凝胶/细胞复合体，并加入培养基进行培养。
进一步地，所述的中性pH下，与缓冲液，例如PBS、DMEM等混合自组装成水凝胶的多肽材料在运用于软骨、血管、神经或皮肤的再生和损伤修复中，具有极大的应用前景。本发明提供的短肽中性条件下与细胞悬液混合后可均匀分散细胞，加入细胞培养基即可进行细胞三维培养，不需要其他操作。
与现有技术相比，本发明具有以下有益效果：
本发明提供了一种可在常温中性pH条件，即在人体生理条件自组装形成三维多孔纳米纤维水凝胶。利用本发明技术制备的材料具有更好的生物相容性，可实现原位负载细胞/活性分子以及体内原位注射，在细胞三维培养、组织工程生物支架材料和药物载体领域具有非常广阔的应用前景和临床应用价值。
附图说明
图1为短肽和Tris-base溶液混合后，再与PBS或DMEM溶液混合，在中性pH条件下，形成水凝胶。
图2为短肽和Tris-base溶液混合后，在其上滴加PBS，在中性pH条件下，形成水凝胶。
图3为在所形成的水凝胶上加入DMEM，渗透到水凝胶的DMEM颜色未改变，说明形成的水凝胶为中性pH。
图4为实施例1中水凝胶材料的原子力纤维镜图。
图5为流变性能对比图，PA/PBS为短肽同PBS混合，PA/DMEM为改性短肽同DMEM混合。G’为储能模量，G”为损耗模量。G’远大于G”，说明形成了稳定的水凝胶。
图6为神经干细胞球在纳米纤维水凝胶中三维生长形貌，从图中可以看出神经干细胞球在本发明专利制备的纳米纤维水凝胶中可以三维生长，并长出较长的轴突。
具体实施方式
下面通过实施例和附图对本发明进行具体描述，有必要在此指出的是本实施例只用于对本发明进行进一步说明，但不能理解为对本发明保护范围的限制，该领域的技术熟练人员可以根据上述本发明的内容作出一些非本质的改进和调整。
除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
短肽的制备：
短肽制备采用通用的制备技术，即多肽合成仪进行合成制备。多肽合成仪以固相合成为反应原理，在密闭的防爆玻璃反应器中使氨基酸按照已知顺序(序列，一般从C端-羧基端 向 N端-氨基端)不断添加、反应、合成，操作最终得到多肽载体。实际合成中，主要采用fmoc固相合成法。
短肽制备：
取一定量的树脂置于固相反应器中，加入5 ml无水 DMF浸泡溶胀2小时，减压抽去溶剂DMF。第一氨基酸耦联到树脂上的方法为，Fmoc保护氨基酸、HBTU和DMAP溶于DMF中，加入DIPEA，室温摇荡反应"10小时，减压抽去溶剂，以DMF洗涤树脂。加入乙酸酐：吡啶：DMF=2：1：3的溶液，摇荡30分钟，封闭树脂上未反应的官能团。依次用DMF，DCM和DMF洗涤树脂，20%派啶/DMF脱保护，时间为10分钟和20分钟。然后按照已知顺序，一般从C端-羧基端向N端-氨基端不断添加、反应、合成，操作最终得到多肽。
多肽的切除和沉淀
树脂用DCM洗涤几遍后，真空抽干。往装有树脂的多肽固相反应器中缓慢加入三氟乙酸/三异丙基硅烷/水溶液，反应3小时.过滤，氮气吹去大部分的溶剂后，向残液中倒入无水乙醚，出现白色絮状沉淀，离心后倒去溶剂乙醚，向沉淀中加入无水乙醚振荡，同样条件下离心，再重复一次，除去大部分的杂质。沉淀真空干燥。固体残留物用离子水溶解，冷冻干燥得到白色絮状固体，-20°C保存。
多肽的分离纯化
将冻干的粗品多肽，溶于20%乙腈/水溶液进行高效液相色谱(HPLC)分离。用含有0.6%的乙腈和水溶液进行梯度洗脱。收集主要峰产物，进行电喷雾质谱或MAL-DI-TOF质谱鉴定。减压旋蒸除去HPLC的样品锋中的乙腈。在冷冻干燥机上冷冻干燥，获得目标产物短肽。
实施例1：水凝胶的多肽材料制备：
将实施例1制备得到的短肽（其序列为:（精氨酸-丙氨酸-天冬氨酸-丙氨酸）4-异亮氨酸-精氨酸-异亮氨酸-精氨酸-异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸）溶于超纯水后，配置成浓度为5~15ml/mL的溶液，与Tris-base（或NaOH）溶液混合，Trsi-base（或NaOH）溶液的浓度为0.1~1 mol/mL，调节其pH值至7，再与PBS（或DMEM）溶液以体积比2：1混合，静置后迅速形成凝胶，待性状稳定后，即可投入使用。
如图1~5所示，混合后形成水凝胶，失去流动性。图4中原子力纤维镜观察水凝胶由纳米纤维构成。
实施例2：三维细胞培养
以超纯水为溶剂，配成浓度为5-15 mg/mL的短肽溶液并和Tris-base（或NaOH）溶液混合，将细胞悬液与调整至中性pH的短肽溶液混合，得到均匀的细胞/短肽混合物，再同PBS（或DMEM）溶液以体积比2:1混合得到包埋有细胞的三维水凝胶，静置后迅速形成凝胶，加入细胞培养基进行培养。
实施例2中包埋细胞为神经干细胞球，如图6所示，可以看出神经干细胞球在本发明专利制备的纳米纤维水凝胶中可以三维生长，并达到了100%的存活率，同时长出较长的轴突。
Claims (3)
1.一种水凝胶材料，其特征在于，将短肽配置成短肽水溶液，先与碱液混合，调节pH得到中性短肽溶液，然后再与缓冲液混合，自组装形成所述水凝胶材料；
所述碱液为NaOH溶液或Tris-base溶液；所述缓冲液为PBS溶液或DMEM溶液；
所述短肽序列为：（精氨酸-丙氨酸-天冬氨酸-丙氨酸）4-异亮氨酸-赖氨酸-异亮氨酸-赖氨酸-异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸；
所述短肽水溶液的浓度为5~15mg/mL；
所述碱液的浓度为0.1~1mol/mL，所述pH为7~7.4；
所述短肽水溶液与缓冲液混合体积比为2：1。
2.权利要求1所述的水凝胶材料在制备组织工程生物支架材料中的应用。
3.权利要求1所述的水凝胶材料在制备神经再生和损伤修复材料中的应用。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105169474A (zh) * | 2015-08-24 | 2015-12-23 | 暨南大学 | 一种中性pH下自组装成水凝胶的多肽材料及其应用 |
| CN105669870A (zh) * | 2016-03-16 | 2016-06-15 | 重庆医科大学附属第一医院 | 自组装多肽d-RADA16-RGD及其制备方法和应用 |
-
2016
- 2016-09-30 CN CN201610868918.7A patent/CN106397545B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105169474A (zh) * | 2015-08-24 | 2015-12-23 | 暨南大学 | 一种中性pH下自组装成水凝胶的多肽材料及其应用 |
| CN105669870A (zh) * | 2016-03-16 | 2016-06-15 | 重庆医科大学附属第一医院 | 自组装多肽d-RADA16-RGD及其制备方法和应用 |
Non-Patent Citations (6)
| Title |
|---|
| Compatibility of Neural Stem Cells with Functionalized Self-assembling Peptide Scaffold in vitro;Zhen Xing Zhang et al.;《Biotechnology and Bioprocess Engineering》;20101231;摘要，第546页Biomaterials * |
| Functional Self-Assembling Peptide Nanofi ber Hydrogels Designed for Nerve Degeneration;Yuqiao Sun et al.;《ACS Applied Materials & Interfaces》;20151231;表1，图2/4，第2353页左栏第1段 * |
| Tuningβ -Sheet Peptide Self-Assembly and Hydrogelation Behavior by Modifica tion of Sequence Hydrophobicity and Aromaticity.;Charles J. Bowerman et al.;《Biomacromolecules》;20110516;摘要，第2740页右栏第4段，图5 * |
| unctional Self-Assembling Peptide Nanofi ber Hydrogels Designed for Nerve Degeneration;Yuqiao Sun et al.;《ACS Applied Materials & Interfaces》;20151231;表1，图2/4，第2353页左栏第1段 * |
| 自组装多肽支架材料的研究进展;王韵晴;《材料科学与工程学报》;20120430;全文 * |
| 自组装多肽纳米纤维水凝胶在细胞三维培养中的应用及特征;刘茜 等;《中国组织工程研究》;20140319;全文 * |
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