Dual-Functionalizable Streptavidin–SpyCatcher-Fused Protein–Polymer Hydrogels as Scaffolds for Cell Culture,ACS Applied Bio Materials

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Dual-Functionalizable Streptavidin–SpyCatcher-Fused Protein–Polymer Hydrogels as Scaffolds for Cell Culture
ACS Applied Bio Materials ( IF 4.6 ) Pub Date : 2020-10-30 , DOI: 10.1021/acsabm.0c00940
Kosuke Minamihata ₁ , Yusei Hamada ₁ , Genki Kagawa ₁ , Wahyu Ramadhan ₁ , Ayato Higuchi ₁ , Kousuke Moriyama ₂ , Rie Wakabayashi ₁ , Masahiro Goto _{1,

3} , Noriho Kamiya _{1,

3}

Affiliation

Hydrogels possessing the ability to control cell functions have great potential as artificial substrates for cell culture. Herein, we report dual-functionalizable protein–polymer hybrid hydrogels prepared by thiol oxidation catalyzed by horseradish peroxidase and a phenolic molecule. A chimera protein of streptavidin (SA) and the SpyCatcher protein, with a cysteine residue at its N-terminus, (C-SA-SC) was constructed and co-cross-linked with thiol-functionalized four-arm polyethylene glycol (PEG-SH) to obtain hydrogels possessing two orthogonal conjugation moieties. Hydrogel formation using C-SA-SC conjugated with biotinylated or SpyTagged functional molecules (premodification strategy) resulted in the formation of hydrogels with a uniform distribution of the functional molecules. Postmodification of the functional molecules of the C-SA-SC hydrogel with biotin or SpyTag could alter the three-dimensional (3D) spatial distribution of the functional molecules within the hydrogels depending on the mode of conjugation (SA/biotin or SpyCatcher/SpyTag), the size of the functional molecules, and the length of time of the modification. NIH-3T3 cells cultured on a C-SA-SC hydrogel, dual-functionalized with a biotinylated-Arg-Gly-Asp-Ser (RGDS) peptide and a basic fibroblast growth factor (bFGF) with SpyTag, showed cell adhesion to the PEG-SH-based hydrogels and cell morphological changes in response to the immobilized RGDS peptide and the bFGF. Moreover, the cells showed higher proliferation on the dual-functionalized C-SA-SC hydrogel than the cells cultured on hydrogels without either the RGDS peptide or the bFGF, demonstrating the benefits of dual-functionalizable hydrogels. The C-SA-SC hydrogel presented in this study is capable of being orthogonally functionalized by two different functional molecules with different 3D distributions of each molecule within the hydrogel and thus has the potential for use as a cell culturing scaffold for creating artificial cellular microstructures.

中文翻译：

双功能链霉亲和素-SpyCatcher-融合蛋白-聚合物水凝胶作为细胞培养的支架

具有控制细胞功能能力的水凝胶具有作为细胞培养的人工基质的巨大潜力。在此，我们报道了由辣根过氧化物酶和酚类分子催化的硫醇氧化制备的双功能化蛋白质-聚合物杂化水凝胶。构建了链霉亲和素 (SA) 和 SpyCatcher 蛋白的嵌合蛋白，在其 N 端具有半胱氨酸残基 (C-SA-SC)，并与硫醇官能化四臂聚乙二醇 (PEG- SH）以获得具有两个正交共轭部分的水凝胶。使用与生物素化或 SpyTagged 功能分子缀合的 C-SA-SC 形成水凝胶（预修饰策略）导致形成具有均匀分布的功能分子的水凝胶。用生物素或 SpyTag 对 C-SA-SC 水凝胶的功能分子进行后修饰可以改变水凝胶内功能分子的三维 (3D) 空间分布，具体取决于缀合模式（SA/生物素或 SpyCatcher/SpyTag），功能分子的大小，以及修饰的时间长度。NIH-3T3 细胞在 C-SA-SC 水凝胶上培养，用生物素化-Arg-Gly-Asp-Ser (RGDS) 肽和带有 SpyTag 的碱性成纤维细胞生长因子 (bFGF) 进行双重功能化，显示细胞粘附到 PEG -SH 基水凝胶和响应固定化 RGDS 肽和 bFGF 的细胞形态变化。此外，与在没有 RGDS 肽或 bFGF 的水凝胶上培养的细胞相比，细胞在双功能化 C-SA-SC 水凝胶上的增殖率更高，展示了双功能化水凝胶的好处。本研究中提出的 C-SA-SC 水凝胶能够被两种不同的功能分子正交功能化，水凝胶中每个分子具有不同的 3D 分布，因此有可能用作细胞培养支架来创建人工细胞微结构。

更新日期：2020-11-16

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