Stiffening of the extracellular matrix is a hallmark in cancer progression, embryonic development, and wound healing. To mimic this dynamic process, our work explored orthogonal enzymatic reactions capable of modulating the properties of poly(ethylene glycol) (PEG)–peptide hydrogels. A hepta-mutant bacterial transpeptidase sortase A (SrtA_7M) was used to ligate two PEG–peptide macromers (i.e., PEG-YLPRTG and NH₂-GGGG-PEG) into a primary hydrogel network. The hydrogels were dynamically stiffened using mushroom tyrosinase (MT), which oxidized tyrosine residues into di-tyrosine and led to increased matrix stiffness. After confirming the expression and enhanced catalytic activity of SrtA_7M, we investigated the cytocompatibility of the enzymatic reaction with a mouse insulinoma cell line, MIN6. In addition, we altered peptide substrate concentrations and evaluated their influence on primary hydrogel network properties and MT-triggered stiffening. Using a pancreatic cancer cell line, COLO-357, the effect of MT-triggered stiffening on spheroid formation was investigated. We found that cell spheroids formed in hydrogels that were exposed to MT were significantly smaller than spheroids formed without MT incubation, suggesting that matrix stiffening played a crucial role in the sizes of cancer cell spheroids. Through utilizing highly specific and orthogonal enzymatic reactions, this hydrogel platform permits rapid and mild in situ cell encapsulation, as well as dynamic control of matrix stiffness for investigating the role of matrix stiffening on cell fate processes.

中文翻译：

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正交酶反应可快速交联和动态调节PEG-肽水凝胶

细胞外基质的强化是癌症进展，胚胎发育和伤口愈合的标志。为了模拟这一动态过程，我们的工作探索了能够调节聚乙二醇（PEG）-肽水凝胶特性的正交酶促反应。使用七突变细菌转肽酶分选酶A（SrtA _7M）将两个PEG肽大分子单体（即PEG-YLPRTG和NH ₂ -GGGG-PEG）连接到一级水凝胶网络中。使用蘑菇酪氨酸酶（MT）使水凝胶动态变硬，后者将酪氨酸残基氧化为二酪氨酸，并导致基质刚度增加。确认SrtA _7M的表达并增强催化活性后，我们研究了与小鼠胰岛素瘤细胞系MIN6进行酶促反应的细胞相容性。此外，我们改变了肽底物的浓度，并评估了它们对主要水凝胶网络性质和MT触发的硬化的影响。使用胰腺癌细胞系COLO-357，研究了MT触发的硬化对球体形成的影响。我们发现暴露于MT的水凝胶中形成的细胞球体明显小于未经MT孵育而形成的球体，这表明基质硬化在癌细胞球体的大小中起着至关重要的作用。通过利用高度特异性和正交的酶促反应，该水凝胶平台可实现快速，温和的原位 细胞封装以及基质刚度的动态控制，以研究基质硬化在细胞命运过程中的作用。