Enhancement of transfection by physical concentration of DNA at the cell surface
Enhancement of transfection by physical concentration of DNA at the cell surface
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DOI：
10.1038/78523
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发表时间：
2000-08-01
影响因子：
46.9
通讯作者：
Saltzman, WM
中科院分区：
文献类型：
--
作者：
Luo, D;Saltzman, WM
高效的DNA转染对于生物学研究和新的临床治疗是至关重要的，但是负责DNA摄取的机制是未知的。目前的非病毒转染方法，经验性地设计，以最大限度地提高DNA络合和/或膜融合，是经得起各种化学品的增强。这些化学品包括微粒、脂质和聚合物复合物，它们优化DNA复合/缩合、膜融合、内体释放或核靶向，这些都是基因递送的假定障碍。大多数化学增强产生适度增加基因传递和有限增加基因表达1。因此，非病毒DNA递送后的转染效率和基因表达水平仍然很低，表明存在其他未识别的障碍。在这里，我们测试的假设，DNA转染效率是有限的一个简单的物理障碍：在细胞表面的低DNA浓度。我们使用致密的二氧化硅纳米颗粒在细胞单层表面浓缩DNA-载体（即DNA-转染试剂）复合物;增加细胞表面复合物浓度的操作使转染效率提高了8.5倍，超过了最好的市售转染试剂。我们预测，旨在优化DNA复合或膜融合的操作有一个基本的物理限制;设计用于提高转染效率的新方法必须在不增加毒性的情况下增加靶细胞表面的DNA浓度。
Efficient DNA transfection is critical for biological research and new clinical therapies, but the mechanisms responsible for DNA uptake are unknown. Current nonviral transfection methods, empirically designed to maximize DNA complexation and/or membrane fusion, are amenable to enhancement by a variety of chemicals. These chemicals include particulates, lipids, and polymer complexes that optimize DNA complexation/condensation, membrane fusion, endosomal release, or nuclear targeting, which are the presumed barriers to gene delivery. Most chemical enhancements produce a moderate increase in gene delivery and a limited increase in gene expression 1. As a result, the efficiency of transfection and level of gene expression after nonviral DNA delivery remain low, suggesting the existence of additional unidentified barriers. Here, we tested the hypothesis that DNA transfection efficiency is limited by a simple physical barrier: low DNA concentration at the cell surface. We used dense silica nanoparticles to concentrate DNA–vector (ie DNA-transfection reagent) complexes at the surface of cell monolayers; manipulations that increased complex concentration at the cell surface enhanced transfection efficiency by up to 8.5-fold over the best commercially available transfection reagents. We predict that manipulations aimed at optimizing DNA complexation or membrane fusion have a fundamental physical limit; new methods designed to increase transfection efficiency must increase DNA concentration at the target cell surface without adding to the toxicity.