全蛋白质水凝胶机械性能的调控及在生物医学中的应用

Hydrogels are 3D networks holding a large amount of water and have received wide applications in biomedical field such as tissue engineering and artificial extracellular matrix. All-protein-based hydrogels are prepared from proteins without additional crosslinkers. Since recombinant proteins are biocompatible and biodegradable and can be prepared with precise sequence, length, composition, structure and biological activity, they are considered as ideal candidates for next-generation biomaterials. The goal of this proposal is to improve the mechanical properties of all-protein-based hydrogels through a synergy between chemical and physical crosslinks. The genetically encoded SpyTag-SpyCatcher protein coupling chemistry will be used to construct a covalent network while the zipping and un-zipping of leucine zippers will serve to dissipate the energy and rebuild strength. Telechilic proteins with SpyTag or SpyCatcher and leucine zipper domains will be designed and prepared. The method will be established to construct a network with both chemical and physical crosslinks. Through judicious site-direct mutagenesis, the role of each crosslinking mechanism in the final material property enhancement will be illustrated. Through systematic variation of the strength, location, and number of leucine zippers in the network, the influence of physical crosslinks on the final hydrogel mechanical properties will be further elucidated and the structure-property relationship will be summarized to guide further optimization of the mechanical properties. Last but not least, we will apply these hydrogels as the artificial extracellular matrix for encapsulating and culturing living cells in three-dimension. We will briefly examine the effects of matrix mechanical properties on the migration, proliferation, and differentiation of the cells.

水凝胶是含有大量水的三维高分子网络，在生物医学领域有着广泛的应用。全蛋白质水凝胶是一种无需外加交联剂的蛋白质基水凝胶，具有优异的生物相容性和可降解性，并能通过基因工程改变相应DNA序列进而精确控制其序列、长度和生物活性，是下一代生物医学的理想材料。本项目以提高全蛋白质水凝胶的机械性能为主要目标，首先基于可基因编码的蛋白质偶联反应对（谍标签SpyTag和谍捕手SpyCatcher）和亮氨酸拉链（Leucine Zipper）的可控聚集，设计并合成可同时发生化学交联和物理交联的反应性蛋白质，建立具有优异机械性能的全蛋白质水凝胶的制备新方法。通过系统改变亮氨酸拉链的强度、位置和数目，全面考察两种交联机理对机械改性的协同作用，总结规律，阐明构效关系以指导进一步优化机械性能。并在此基础上利用目标水凝胶封装和培养活细胞，初步考察不同的力学环境对于细胞生存、迁移、繁衍和分化等行为的影响。

本项目旨在发展可在基因层次上编码其宏观性质的全蛋白质水凝胶，并探索其生物医药应用。为了实现化学交联和物理交联的协同，开发了具有各种特性的可基因编码的蛋白质偶联反应对的工具家族，以实现对化学交联更好的控制，并结合“组装-反应”协同的理念成功制备了包括蛋白质索烃、星状蛋白等一系列拓扑蛋白质，为蛋白质水凝胶提供了独特的结构基元，在此基础上，进一步研究了控制基于蛋白-蛋白相互作用的物理交联（包括拓扑缠结、聚集、静电相互作用等）的若干方法，初步实现了具有可控机械性能和良好生物相容性的全蛋白质水凝胶，可应用于封装和培养活细胞，有望在组织工程领域得到广泛的应用。此外，基于彼此正交的多肽-蛋白质化学反应对，我们应用使用层层组装的方法将全蛋白质水凝胶网络的维度降低，成为二维的功能薄膜，实现了对于不同基底的功能化，并发现网络中功能蛋白的活性和稳定性随着层数增加均有所提升。总的来说，在本项目资助下，我们做出了一批富有特色的原创性工作，提出蛋白质拓扑工程学的研究方向，申请了两个专利，并有一个已经得到授权，已在国内外知名学术期刊上发表了23篇论文，产生了重要的学术影响。