Department of Biological Systems Engineering, University of Nebraska-Lincoln, Lincoln, NE, 68583, USA.
B CUBE - Center for Molecular Bioengineering, Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Tatzberg 41, Dresden 01307, Germany.
Exp Biol Med (Maywood). 2020 Apr;245(7):606-619. doi: 10.1177/1535370220913501. Epub 2020 Mar 17.
Nonviral gene delivery, though limited by inefficiency, has extensive utility in cell therapy, tissue engineering, and diagnostics. Substrate-mediated gene delivery (SMD) increases efficiency and allows transfection at a cell-biomaterial interface, by immobilizing and concentrating nucleic acid complexes on a surface. Efficient SMD generally requires substrates to be coated with serum or other protein coatings to mediate nucleic acid complex immobilization, as well as cell adhesion and growth; however, this strategy limits reproducibility and may be difficult to translate for clinical applications. As an alternative, we screened a chemically defined combinatorial library of 20 different extracellular matrix mimetic substrates containing combinations of (1) different sulfated polysaccharides that are essential extracellular matrix glycosaminoglycans (GAGs), with (2) mimetic peptides derived from adhesion proteins, growth factors, and cell-penetrating domains, for use as SMD coatings. We identified optimal substrates for DNA lipoplex and polyplex SMD transfection of fibroblasts and human mesenchymal stem cells. Optimal extracellular matrix mimetic substrates varied between cell type, donor source, and transfection reagent, but typically contained Heparin GAG and an adhesion peptide. Multiple substrates significantly increased transgene expression (i.e. 2- to 20-fold) over standard protein coatings. Considering previous research of similar ligands, we hypothesize extracellular matrix mimetic substrates modulate cell adhesion, proliferation, and survival, as well as plasmid internalization and trafficking. Our results demonstrate the utility of screening combinatorial extracellular matrix mimetic substrates for optimal SMD transfection towards application- and patient-specific technologies.
Substrate-mediated gene delivery (SMD) approaches have potential for modification of cells in applications where a cell-material interface exists. Conventional SMD uses ill-defined serum or protein coatings to facilitate immobilization of nucleic acid complexes, cell attachment, and subsequent transfection, which limits reproducibility and clinical utility. As an alternative, we screened a defined library of extracellular matrix mimetic substrates containing combinations of different glycosaminoglycans and bioactive peptides to identify optimal substrates for SMD transfection of fibroblasts and human mesenchymal stem cells. This strategy could be utilized to develop substrates for specific SMD applications in which variability exists between different cell types and patient samples.
非病毒基因传递，尽管效率有限，但在细胞治疗、组织工程和诊断学中具有广泛的应用。基底介导的基因传递（SMD）通过将核酸复合物固定在表面上，增加了效率，并允许在细胞-生物材料界面处进行转染。高效的 SMD 通常需要将底物用血清或其他蛋白质涂层覆盖，以介导核酸复合物的固定，以及细胞的附着和生长；然而，这种策略限制了可重复性，并且可能难以转化为临床应用。作为替代方案，我们筛选了一个由 20 种不同的细胞外基质模拟底物组成的化学定义组合文库，这些底物包含（1）不同的硫酸化多糖，它们是必需的细胞外基质糖胺聚糖（GAGs），与（2）源自粘附蛋白、生长因子和细胞穿透结构域的模拟肽。我们确定了用于 DNA 脂质体和多聚物 SMD 转染成纤维细胞和人间充质干细胞的最佳底物。最佳的细胞外基质模拟底物因细胞类型、供体来源和转染试剂而异，但通常包含肝素 GAG 和粘附肽。多种底物显著增加了转基因表达（即 2 到 20 倍），超过了标准蛋白质涂层。考虑到类似配体的先前研究，我们假设细胞外基质模拟底物调节细胞的附着、增殖和存活，以及质粒的内化和运输。我们的结果表明，筛选组合细胞外基质模拟底物对于针对特定应用和患者的技术进行最佳 SMD 转染具有实用性。
基底介导的基因传递（SMD）方法有可能在存在细胞-材料界面的应用中修饰细胞。传统的 SMD 使用定义不明确的血清或蛋白质涂层来促进核酸复合物的固定、细胞附着和随后的转染，这限制了可重复性和临床实用性。作为替代方案，我们筛选了一个包含不同糖胺聚糖和生物活性肽组合的细胞外基质模拟底物的定义文库，以确定用于成纤维细胞和人间充质干细胞 SMD 转染的最佳底物。这种策略可以用于开发特定 SMD 应用的底物，在这些应用中，不同细胞类型和患者样本之间存在可变性。
