Department of Chemistry, Macromolecular Science and Engineering Program, and Michigan Nanotechnology Institute for Medicine and Biological Sciences (MNIMBS), University of Michigan, 911 North University Avenue, Ann Arbor, MI 48109-1055, USA.
Mol Pharm. 2010 Jun 7;7(3):870-83. doi: 10.1021/mp100027g.
Polycationic materials commonly used to delivery DNA to cells are known to induce cell membrane porosity in a charge-density dependent manner. It has been suggested that these pores may provide a mode of entry of the polymer-DNA complexes (polyplexes) into cells. To examine the correlation between membrane permeability and biological activity, we used two-color flow cytometry on two mammalian cell lines to simultaneously measure gene expression of a plasmid DNA delivered with four common nonviral vectors and cellular uptake of normally excluded fluorescent dye molecules of two different sizes, 668 Da and 2 MDa. We also followed gene expression in cells sorted based on the retention of endogenous fluorescein. We have found that cell membrane porosity caused by polycationic vectors does not enhance internalization or gene expression. Based on this single-cell study, membrane permeability is found to be an unwanted side effect that limits transfection efficiency, possibly through leakage of the delivered nucleic acid through the pores prior to transcription and translation and/or activation of cell defense mechanisms that restrict transgene expression.
常用的将 DNA 递送至细胞的聚阳离子材料以电荷密度依赖的方式诱导细胞膜通透性。据认为，这些孔可能为聚合物-DNA 复合物（多聚物）进入细胞提供了一种方式。为了检查细胞膜通透性与生物活性之间的相关性，我们使用双色流式细胞术在两种哺乳动物细胞系上同时测量了用四种常见的非病毒载体递送的质粒 DNA 的基因表达和通常被排斥的两种不同大小的荧光染料分子（668 Da 和 2 MDa）的细胞内摄取。我们还跟踪了基于内源性荧光素保留的细胞分选后的基因表达。我们发现聚阳离子载体引起的细胞膜通透性不会增强内化或基因表达。基于这项单细胞研究，发现细胞膜通透性是一种不受欢迎的副作用，它通过在转录和翻译之前通过孔泄漏递送至的核酸和/或激活限制转基因表达的细胞防御机制来限制转染效率。
