Zhou Ping, Brown Lauren, Madl Christopher M
Department of Materials Science & Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
Adv Funct Mater. 2025 May 30. doi: 10.1002/adfm.202422047.
Hydrogels are routinely used as scaffolds to mimic the extracellular matrix for tissue engineering. However, common strategies to covalently crosslink hydrogels employ reaction conditions with potential off-target biological reactivity. The limited number of suitable bioorthogonal chemistries for hydrogel crosslinking restricts how many material properties can be independently addressed to control cell fate. To expand the bioorthogonal toolkit available for hydrogel crosslinking, we identify isonitrile ligations as a promising class of reactions. Isonitriles are compact, stable, selective, and biocompatible moieties that react with chlorooxime (ChO), tetrazine (Tz), and azomethine imine (AMI) functional groups under physiological conditions. We demonstrate that all three ligation reactions can form hydrogels, with isonitrile-ChO ligation exhibiting optimal gelation properties. Synthetic poly(ethylene glycol) (PEG) hydrogels crosslinked by isonitrile-ChO ligation exhibit rapid gelation kinetics, elastic mechanical properties, stability under physiological conditions, and high biocompatibility. By combining ChO-functionalized multi-arm PEGs with isonitrile-functionalized engineered elastin-like proteins (ELPs), we demonstrate simultaneous control over network connectivity and adhesive ligand presentation, which in turn regulate cell spreading. These hydrogels enable the long-term culture of numerous human cell types relevant to regenerative medicine. Furthermore, we demonstrate that isonitrile-ChO ligation is orthogonal to common azide-alkyne cycloaddition, enabling independent, bioorthogonal functionalization of hydrogels containing live cells.
水凝胶通常被用作支架来模拟细胞外基质以用于组织工程。然而，将水凝胶共价交联的常见策略所采用的反应条件具有潜在的脱靶生物反应性。适用于水凝胶交联的生物正交化学方法数量有限，限制了可独立调控以控制细胞命运的材料特性的数量。为了扩展可用于水凝胶交联的生物正交工具包，我们确定异腈连接反应是一类有前景的反应。异腈是紧凑、稳定、具有选择性且生物相容的基团，在生理条件下可与氯肟（ChO）、四嗪（Tz）和甲亚胺亚胺（AMI）官能团发生反应。我们证明所有这三种连接反应都能形成水凝胶，其中异腈 - ChO连接表现出最佳的凝胶化特性。通过异腈 - ChO连接交联的合成聚乙二醇（PEG）水凝胶具有快速的凝胶化动力学、弹性力学性能、在生理条件下的稳定性以及高生物相容性。通过将ChO功能化的多臂PEG与异腈功能化的工程化类弹性蛋白（ELP）相结合，我们展示了对网络连通性和黏附配体呈现的同时控制，进而调节细胞铺展。这些水凝胶能够对与再生医学相关的多种人类细胞类型进行长期培养。此外，我们证明异腈 - ChO连接与常见的叠氮化物 - 炔烃环加成反应正交，能够对含有活细胞的水凝胶进行独立的、生物正交的功能化修饰。
