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Screening a chemically defined extracellular matrix mimetic substrate library to identify substrates that enhance substrate-mediated transfection.
Experimental Biology and Medicine ( IF 3.2 ) Pub Date : 2020-03-17 , DOI: 10.1177/1535370220913501
Andrew Hamann 1 , Alvin K Thomas 2 , Tyler Kozisek 1 , Eric Farris 1 , Steffen Lück 2 , Yixin Zhang 2 , Angela K Pannier 1
Affiliation  

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.

Impact statement

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.



中文翻译:

筛选化学定义的细胞外基质模拟底物文库,以鉴定增强底物介导的转染的底物。

非病毒基因传递尽管受到效率低下的限制,但在细胞治疗,组织工程和诊断中具有广泛的用途。通过将核酸复合物固定并浓缩在表面上,底物介导的基因传递(SMD)可提高效率并允许在细胞-生物材料界面进行转染。高效的SMD通常需要在基质上涂上血清或其他蛋白质涂层,以介导核酸复合物的固定以及细胞粘附和生长。但是,这种策略限制了可重复性,可能难以转化为临床应用。作为备选,我们筛选了20种不同的细胞外基质模拟物底物的化学定义组合文库,这些底物包含(1)是必需的细胞外基质糖胺聚糖(GAG)的不同硫酸化多糖与(2)来源于粘附蛋白,生长因子和细胞分子的模拟肽的组合渗透域,用作SMD涂料。我们确定了成纤维细胞和人间充质干细胞的DNA脂质复合物和多聚体SMD转染的最佳底物。最佳的细胞外基质模拟底物在细胞类型,供体来源和转染试剂之间有所不同,但通常包含肝素GAG和粘附肽。与标准蛋白质涂层相比,多种底物显着提高了转基因表达(即2至20倍)。考虑到以前对类似配体的研究,我们假设细胞外基质模拟底物调节细胞粘附,增殖和生存,以及质粒内在化和运输。我们的结果证明了筛选组合细胞外基质模拟底物对于针对应用和患者特定技术进行最佳SMD转染的实用性。

影响陈述

在存在细胞-材料界面的应用中,底物介导的基因传递(SMD)方法具有修饰细胞的潜力。常规的SMD使用不确定的血清或蛋白涂层来促进核酸复合物的固定,细胞附着以及随后的转染,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库,以鉴定成纤维细胞和人间充质干细胞SMD转染的最佳底物。该策略可用于开发特定SMD应用的底物,其中不同细胞类型和患者样品之间存在差异。

更新日期:2020-04-10
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