Department of Chemistry and ‡Stem Cell Institute and Division of Pediatric Blood and Marrow Transplantation, University of Minnesota , Minneapolis, Minnesota, United States.
Biomacromolecules. 2017 Jan 9;18(1):56-67. doi: 10.1021/acs.biomac.6b01297. Epub 2016 Dec 19.
Improving the delivery of nucleic acids to diverse tissue types in culture is important for translating genome editing for regenerative cell therapies. Herein, we examine the effect of transfection media additives, such as the sulfated glycosaminoglycan heparin, in dramatically increasing pDNA delivery efficiency and transgene expression in a wide variety of cell types. Polyplexes formed by combining pDNA and Tr4, a cationic glycopolymer containing repeated trehalose and pentaethylenetetramine groups, were treated with low concentrations of heparin prior to in vitro transfection with plasmid DNA. Polyplex formulations were found to be stable and form ternary complexes upon heparin addition according to dynamic light scattering and ethidium bromide dye exclusion assays. Heparin-coated polyplexes offer significant increases (approximately 4-fold) in GFP expression compared to polyplexes prepared with Tr4 only in primary fibroblasts, U87, and HepG2 cells. Heparin was also shown to increase GFP expression in a linear, dose-dependent manner. The heparin-treated Tr4 polyplexes exhibited more than 50% higher cellular internalization with HepG2 cells while showing minimal increases with U87 and primary fibroblasts. Pharmacological inhibition was used to further understand the endocytic pathways taken during transfection in the presence and absence of heparin. It was found that heparin-treated polyplexes are endocytosed primarily through macropinocytosis and clathrin-mediated pathways, while Tr4 polyplexes without heparin appear to be internalized primarily via caveolae. Heparin appears to also modify the nuclear localization behavior of Tr4 polyplexes, which likely contributes to increased efficiency and transgene expression.
提高基因编辑在再生细胞疗法中的应用效率，关键在于将核酸递送到不同组织类型的细胞中。本文研究了转染培养基添加剂（如硫酸化糖胺聚糖肝素）对提高多种细胞类型中 pDNA 转染效率和转基因表达的影响。在体外转染质粒 DNA 之前，先用低浓度肝素处理由 pDNA 和含有重复海藻糖和五亚乙基六胺基团的阳离子糖聚合物 Tr4 形成的多聚物。根据动态光散射和溴化乙锭染料排除实验，发现多聚物制剂在添加肝素后是稳定的，并形成三元复合物。与仅用 Tr4 制备的多聚物相比，肝素包被的多聚物在原代成纤维细胞、U87 和 HepG2 细胞中显著提高 GFP 表达（约 4 倍）。肝素还以线性、剂量依赖的方式增加 GFP 表达。肝素处理的 Tr4 多聚物与 HepG2 细胞的细胞内摄取率提高了 50%以上，而与 U87 和原代成纤维细胞的增加率最小。本文还使用药理学抑制来进一步了解在有或没有肝素的情况下转染过程中采用的内吞途径。结果发现，肝素处理的多聚物主要通过巨胞饮作用和网格蛋白介导的途径内吞，而没有肝素的 Tr4 多聚物似乎主要通过胞吞作用内吞。肝素似乎还改变了 Tr4 多聚物的核定位行为，这可能有助于提高效率和转基因表达。
