Guo Chen, Kim Heejae, Ovadia Elisa M, Mourafetis Christine M, Yang Mingrui, Chen Wilfred, Kloxin April M
Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716, United States.
Department of Chemical and Biomolecular Engineering, New York University, Brooklyn, NY 11201, United States.
Acta Biomater. 2017 Jul 1;56:80-90. doi: 10.1016/j.actbio.2017.04.002. Epub 2017 Apr 5.
Hydrogels are facile architectures for the controlled presentation of proteins with far-reaching applications, from fundamental biological studies in three-dimensional culture to new regenerative medicine and therapeutic delivery strategies. Here, we demonstrate a versatile approach for spatially-defined presentation of engineered proteins within hydrogels through i) immobilization using bio-orthogonal strain-promoted alkyne-azide click chemistry and ii) dynamic protease-driven protein release using exogenously applied enzyme. Model fluorescent proteins were expressed using nonsense codon replacement to incorporate azide-containing unnatural amino acids in a site-specific manner toward maintaining protein activity: here, cyan fluorescent protein (AzCFP), mCherry fluorescent protein (AzmCh), and mCh decorated with a thrombin cut-site. (AzTMBmCh). Eight-arm poly(ethylene glycol) (PEG) was modified with dibenzylcyclooctyne (DBCO) groups and reacted with azide functionalized PEG in aqueous solution for rapid formation of hydrogels. Azide functionalized full-length fluorescent proteins were successfully incorporated into the hydrogel network by reaction with PEG-DBCO prior to gel formation. Temporal release and removal of select proteins (AzTMBmCh) was triggered with the application of thrombin and monitored in real-time with confocal microscopy, providing a responsive handle for controlling matrix properties. Hydrogels with regions of different protein compositions were created using a layering technique with thicknesses of hundreds of micrometers, affording opportunities for the creation of complex geometries on size scales relevant for controlling cellular microenvironments.
Controlling protein presentation within biomaterials is important for modulating interactions with biological systems. For example, native tissues are composed of subunits with different matrix compositions (proteins, stiffness) that dynamically interact with cells, influencing function and fate. Toward mimicking such temporally-regulated and spatially-defined microenvironments, we utilize bio-orthogonal click chemistry and protein engineering to create hydrogels with distinct regions of proteins and modify them over time. Through nonsense codon replacement, we site-specifically functionalize large proteins with i) azides for covalent conjugation and ii) an enzymatic cleavage site for user-defined release from hydrogels. Our results exemplify not only the ability to create unique bio-functionalized hydrogels with controlled mechanical properties, but also the potential for creating interesting interfaces for cell culture and tissue engineering applications.
水凝胶是用于可控呈现蛋白质的简便结构，具有广泛的应用，从三维培养中的基础生物学研究到新的再生医学和治疗递送策略。在此，我们展示了一种通用方法，用于在水凝胶中对工程蛋白进行空间定义的呈现，方法包括：i）使用生物正交应变促进的炔烃 - 叠氮化物点击化学进行固定，以及ii）使用外源施加的酶进行动态蛋白酶驱动的蛋白释放。使用无义密码子替换表达模型荧光蛋白，以位点特异性方式掺入含叠氮化物的非天然氨基酸以维持蛋白活性：在此，青色荧光蛋白（AzCFP）、mCherry荧光蛋白（AzmCh）以及带有凝血酶切割位点修饰的mCh（AzTMBmCh）。八臂聚乙二醇（PEG）用二苄基环辛炔（DBCO）基团修饰，并在水溶液中与叠氮化物功能化的PEG反应以快速形成水凝胶。在凝胶形成之前，通过与PEG - DBCO反应，将叠氮化物功能化的全长荧光蛋白成功掺入水凝胶网络。通过施加凝血酶触发特定蛋白（AzTMBmCh）的时间释放和去除，并使用共聚焦显微镜实时监测，为控制基质特性提供了一个响应性手段。使用分层技术创建具有不同蛋白质组成区域的水凝胶，其厚度为数百微米，为在与控制细胞微环境相关的尺寸尺度上创建复杂几何形状提供了机会。
控制生物材料内的蛋白质呈现对于调节与生物系统的相互作用很重要。例如，天然组织由具有不同基质组成（蛋白质、硬度）的亚基组成，这些亚基与细胞动态相互作用，影响功能和命运。为了模拟这种时间调节和空间定义的微环境，我们利用生物正交点击化学和蛋白质工程来创建具有不同蛋白质区域的水凝胶，并随时间对其进行修饰。通过无义密码子替换，我们用i）用于共价结合的叠氮化物和ii）用于从水凝胶中用户定义释放的酶切位点对大蛋白进行位点特异性功能化。我们的结果不仅例证了创建具有可控机械性能的独特生物功能化水凝胶的能力，还例证了为细胞培养和组织工程应用创建有趣界面的潜力。
