环境敏感型beta-发夹状自组装短肽的设计及水凝胶生物学性能研究

Scaffold development has become an important area in tissue engineering and reparative and regenerative medicine. Recently, a few peptide-based self-assembling systems have been explored as useful nanobiomaterials in applications such as clinical trials, 3D cell culture and tissue engineering because of their extreme purity and excellent function and biocompatibility. However, it is still a challenge for scientists and engineers to incorporate bioactive elements into systematic design of the new generation of smart and multifunctional nanohydrogel materials with specialized inherent bioactivities for clinical use in tissue engineering. In our previous work, two molecules of antimicrobial peptide sequences (KIGAKI)3 were connected with a tetrapeptide linker Thr-DPro-Pro-Gly which served as a centrically loop. When exposed to stimuli such as pH, ionic and heat, this peptide was capable of undergoing a reversible transition from random coil to beta-hairpin structures and further self-assembled into high-aspect-ratio nanofibers and afforded the hydrogel whose surface is inherently antibacterial. In order to further improve our results and enhance our understanding of the fabrication of peptide-based materials, bioactive sequences such as IKVAV, LRGDV and KIGAKI are chosen here as basic building elements to create new stimuli-responsive self-assembling peptides. In this proposal, the structure conversion, structural properties, self-assembly kinetics of the designer peptides and rheological properties of the hydrogels will be firstly characterized. After these steps, to lay a foundation for further theoretical research and effective application of the bioactive sequences comprising peptide hydrogels in tissue engineering, the in vitro toxicology, cytocompatibility and inherent bioactivities of the hydrogel networks will be studied. In summary, this research will provide a new approach for the design of smart, self-assembling nanohydrogels with inherent bioactivities by using bioactive peptide sequences as basic building blocks. And this series of designer peptide may find use in 3D cell culture and in tissue engineering and regenerative medicine as hydrogel scaffolds to support tissue formation in the future.

支架材料研究是组织工程和再生医学的重要领域。短肽自组装材料因具有极高的纯度、良好的组织相容性已用于临床、细胞三维培养和组织工程研究。但将多种生物活性元素融入短肽材料中，设计新一代具有特化功能的"智能"纳米水凝胶材料还是一个颇具挑战性的课题。我们以前的研究将T(DP)PG 四肽作为中心转角连接两分子抗菌肽序列(KIGAKI)3，获得了能够响应pH、盐离子和温度变化的短肽，并自组装形成纳米纤维和具有抗菌活性的水凝胶。本课题主要拟选择IKVAV 、LRGDV和KIGAKI 三个活性序列作为短肽基本构建单元来设计新型"智能"自组装短肽水凝胶，研究短肽的二级结构转变、纤维结构特征、自组装动力学和水凝胶流变特征等，进而研究水凝胶的体外毒性和细胞相容性及可能具备的固有生物活性。这类水凝胶材料的诞生，不但从赋予特异活性方面极大丰富了自组装短肽材料的设计思路，更为细胞治疗和组织工程提供了特定功能的载体材料。

至今已有许多自组装短肽系统被设计出，它们代表了小分子工程上的重要进展，已被广泛应用于诸如药物缓释、三维培养、再生医学、生物传感器、表面技术和纳米工程等生物医学和纳米科技领域。目前发现和设计生物来源新材料仍是焦点。在前期研究工作的基础上，我们进行了以下几方面的研究：(1) 用IKVAV、LRGDV和KIGAKI等生物活性序列作为基本构建单元设计新型自组装短肽。筛选发现了一些可能在生理条件下形成水凝胶的短肽序列，可以有效支持人羊膜间充质干细胞的增殖和分化；(2) 利用弹性蛋白序列设计自组装短肽序列，研究发现疏水作用、分子内静电作用和链熵之间的平衡决定短肽分子状态和自组装。水凝胶中对bFGF的包裹不会影响其二级结构和生物活性。bFGF的缓释能有效促进NIH-3T3细胞的增殖，并激活ERK等下游通路；(3) 我们用生物素夹心法将小分子蛋白质IGF-1绑定到短肽纤维上从而达到缓释的效果，并可以有效促进ATDC5细胞增殖；(4) 我们设计了富组氨酸的功能化自组装短肽序列用来指导纳米颗粒的定位组装。短肽在中性(pH 7.2)时能够自组装成纳米纤维，并在酸性(pH 3.8)中在肽骨架的方向上通过纳米纤维的层次组装形成纳米膜。短肽可以作为金晶体成核和生长的模板。实验表明金晶体能够在纤维两侧生长，并分别在中性和酸性条件下形成一维的轨道状结构和二维膜。金晶体的大小和密度和金前体孵育时间正相关。我们的方法可以用于金属、金属硫化物和金属氧化物一维和二维结构的可控合成，并且费用更加低廉，方法更加简单。此外，它们可以有效催化对硝基苯酚水解，并可至少重复使用三次而没有明显活性损失；(5) 我们设计了一系列含有咪唑基的自组装短肽，可以催化2,4-二硝基苯甲酸和对硝基苯酯水解。短肽催化反应动力学符合米氏方程。同时短肽纤维表现出对具有短脂肪酸链底物的特异性，这和自然酯酶类似。竞争性抑制实验表明纤维beta折叠片疏水表面和底物分子之间的相互作用在底物识别中起到重要作用。