基于酶促反应的短肽自组装水凝胶构筑、性能调控及其在细胞培养中的应用

Due to cellular origin, fibrillar hydrogels formed via peptide self-assembly have shown great potential as scaffolds in cell culture and regenerative medicine. Despite their biocompatibility, several drawbacks are inherently associated with the self-assembling peptide hydrogels, i.e., 1) they are generally relative weak and soft, thus being unable to provide sufficient mechanical support and withstand medium change as scaffolds in some biological applications; 2) limited cell-binding motifs are on their surface, thus being unable to elicit more cellular responses; 3) they are typically stable and resistant to proteolysis, due to lack of specific enzymatic cleavable sites as well as their amyloid-like structure. To overcome these problems and simultaneously avoid the disadvantages caused by chemical cross-linking and complicated co-assembly of peptides and their bio-motif bearing derivatives, we turn to enzymatic reactions that are ubiquitous in biology. It is well known that enzymes work at mild conditions and enzymatic reactions are highly specific and efficient. In this proposal, we will take these advantages to control mechanical and biological performances of peptide self-assembling hydrogels. We will focus on our efforts on: 1) the design of self-assembling short amphiphilic peptides with certain residues or sequences that can be specifically recognized by transglutaminase (TG), lysyl oxidase (LO) or plasma amine oxidase (PAO), and matrix metalloproteinase (MMP); 2) the utilization of enzymatic reactions for surface biofunctionalization with bioactive ligands (e.g. RGDS) and cross-linking of self-assembled nanofibers, and enzymatic degradation of monomers and their different hierarchical assemblies; 3) 2D and 3D cell culture with the resultant peptide self-assembling hydrogels as scaffolds, the effect of their mechanical strength, biofunctionality, and degradability on cell adhesion, spread, migration, proliferation, and differentiation, and non-specific immunogenic responses induced by these gels. We expect that these investigations will favor the development of novel approaches for the fabrication of multiple-functional assemblies via hierarchical processes and peptide self-assembling scaffolds with close structural and biochemical similarity to the natural extracellular matrix (ECM).

肽自组装纳米纤维水凝胶作为支架材料在再生医学中展现出良好的应用前景，但存在机械强度低、与细胞相互作用能力弱、生物降解可控性差等问题。酶促反应在机体内普遍存在，具有反应条件温和、特异性强且转化率高等特点，本项目拟利用其来调控肽自组装水凝胶的机械性能和生物功能。研究主要包括：1）构建新颖的自组装短肽，分子中含有谷氨酰胺转胺酶、赖氨酰氧化酶、基质金属蛋白酶等功能酶的底物氨基酸残基或序列；2）将自组装与酶促反应相结合，通过后者将自组装纳米纤维表面功能化和共价交联，发展多层次、分级构筑超分子功能体的新方法，获得多功能集成的肽水凝胶体系；3）将肽水凝胶作为支架材料用于细胞的二维和三维培养，研究其机械性能、配体功能化和酶解性能对细胞生长和分化的影响，并评价材料的免疫反应。项目设计可以避免化学交联、共组装等手段带来的不利影响，发展可控构筑组装体的新方法，赋予组装体更多的生物功能，促进其在生物医学中的应用。

酶在生物体内普遍存在，酶促反应具有反应条件温和、催化效率高、专一性强等特点。肽分子自组装可以形成规则有序的纳米聚集体，这些聚集体具有特定的物理化学性能和生物学功能。此外，特定氨基酸残基或序列通常为酶的作用底物。为获得生物响应性分子和材料，本项目将酶促反应和肽自组装相结合，在深入理解肽自组装机制和规律的基础上，来构筑酶响应性自组装体系或材料。主要的研究内容和重要的结果包括：1）设计了谷氨酰胺转移酶TGase响应的Ac-I3QGK-NH2分子，在TGase作用下可以自组装形成水凝胶并用于快速、有效止血；2）通过TGase将Ac-I4K2-NH2自组装纳米纤维表面功能化，来诱导细胞的粘附和迁移；3）抗菌肽Ac-A9K2-NH2在血浆氧化酶PAO或赖氨酸氧化酶LO作用下自组装成水凝胶，该水凝胶展现出良好的抗菌性能和细胞相容性，作为细胞培养的支架材料可以有效防止细菌污染；4）设计了Ac-I3SLKG-NH2分子，其自组装所形成的水凝胶可以被金属蛋白酶MMP-2有效降解；5）设计了Ac-I4K2Y*-NH2分子，对Fe(III)或酪氨酸酶响应成胶，可以选择性杀死小鼠黑色素瘤细胞(B16)，而对其他癌细胞和正常细胞毒性低；6）通过设计其他两亲性短肽、hairpin肽、离子互补短肽等，深入理解了分子手性与组装体手性、组装过程中的手性跨尺度传递、肽自组装分级过程和驱动力等科学问题，初步实现了肽自组装纳米结构和性能的调控；7）设计了系列抗菌、抗肿瘤螺旋肽，这些肽具有非常好的细胞选择性、生物相容性，且多次使用后不会导致病菌产生抗性。该项目把酶作用底物氨基酸或序列引入到肽自组装基元分子中，赋予了肽自组装更多的可控性能和生物功能，将大大拓展其在生物医学中的应用。

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