Department of Biomedical Engineering, Purdue School of Engineering & Technology, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA.
Biomater Sci. 2017 Oct 24;5(11):2231-2240. doi: 10.1039/c7bm00691h.
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) was used to ligate two PEG-peptide macromers (i.e., PEG-YLPRTG and NH-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, 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.
细胞外基质的僵硬是癌症进展、胚胎发育和伤口愈合的一个标志。为了模拟这个动态过程，我们的工作探索了能够调节聚乙二醇（PEG）-肽水凝胶性质的正交酶反应。使用七突变细菌转肽酶 SrtA 将两种 PEG-肽大分子（即 PEG-YLPRTG 和 NH-GGGG-PEG）连接成初级水凝胶网络。使用蘑菇酪氨酸酶（MT）动态增强水凝胶的硬度，MT 将酪氨酸残基氧化成二酪氨酸，导致基质硬度增加。在确认 SrtA 的表达和增强的催化活性后，我们用小鼠胰岛素瘤细胞系 MIN6 研究了酶反应的细胞相容性。此外，我们改变了肽底物浓度，并评估了它们对初级水凝胶网络性质和 MT 引发的硬度增加的影响。使用胰腺癌细胞系 COLO-357，研究了 MT 引发的硬度增加对球体形成的影响。我们发现，暴露于 MT 的水凝胶中形成的细胞球体明显小于没有 MT 孵育形成的球体，这表明基质硬度在癌细胞球体的大小中起着关键作用。通过利用高度特异性和正交的酶反应，这个水凝胶平台允许快速和温和的原位细胞封装，以及基质硬度的动态控制，以研究基质硬度对细胞命运过程的作用。
