Mechanisms of cellular uptake and intracellular trafficking with chitosan/DNA/poly(γ-glutamic acid) complexes as a gene delivery vector
Mechanisms of cellular uptake and intracellular trafficking with chitosan/DNA/poly(γ-glutamic acid) complexes as a gene delivery vector
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DOI：
10.1016/j.biomaterials.2010.08.081
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发表时间：
2011-01-01
期刊：
影响因子：
14
通讯作者：
Sung, Hsing-Wen
中科院分区：
文献类型：
--
作者：
Peng, Shu-Fen;Tseng, Michael T.;Sung, Hsing-Wen
壳聚糖(CS)络合物被认为是一种DNA载体，但其转染率相对较低。在我们先前的研究中，我们发展了一种通过在CS/DNA复合体中掺入聚γ-谷氨酸(Gamma-PGA)来提高其基因表达水平的方法：然而，具体的机制仍不清楚。本研究旨在探讨CS/DNA/γ-PGA复合体在细胞内摄取和转运的机制。分子动力学模拟的结果表明，γ-PGA与CS形成复合体后，在其N端显示一个游离的γ-谷氨酸，从而可能被细胞膜上的γ-谷氨酰转肽酶识别，导致细胞摄取显著增加。在内吞抑制研究中，我们发现CS/DNA复合体的内化是通过巨噬细胞吞噬和小凹介导的途径进行的；通过在复合体中掺入γ-PGA，这两条摄取途径都得到了进一步的增强，但小凹介导的途径起主要作用。用透射电子显微镜直接了解了测试络合物的内化机理，并证实了我们在缓蚀实验中的发现。内化后，与CS/DNA相比，CS/DNA/γ-PGA复合体与溶酶体共定位的百分比较低。细胞摄取的增加和进入溶酶体的减少可能解释了CS/DNA/γ-PGA复合体的转染率提高的原因。了解这些涉及含有γ-PGA的壳聚糖基复合体的机制对于开发有效的DNA转移载体至关重要。(C)2010爱思唯尔有限公司。保留所有权利。
Chitosan (CS)-based complexes have been considered as a vector for DNA delivery; nonetheless, their transfection efficiency is relatively low. An approach by incorporating poly(gamma-glutamic acid) (gamma-PGA) in CS/DNA complexes was developed in our previous study to enhance their gene expression level: however, the detailed mechanisms remain to be understood. The study was designed to investigate the mechanisms in cellular uptake and intracellular trafficking of CS/DNA/gamma-PGA complexes. The results of our molecular dynamic simulations suggest that after forming complexes with CS, gamma-PGA displays a free gamma-glutamic acid in its N-terminal end and thus may be recognized by gamma-glutamyl transpeptidase in the cell membrane, resulting in a significant increase in their cellular uptake. In the endocytosis inhibition study, we found that the internalization of CS/DNA complexes took place via macropinocytosis and caveolae-mediated pathway; by incorporating gamma-PGA in complexes, both uptake pathways were further enhanced but the caveolae-mediated pathway played a major role. TEM was used to gain directly understanding of the internalization mechanism of test complexes and confirmed our findings obtained in the inhibition experiments. After internalization, a less percentage of co-localization of CS/DNA/gamma-PGA complexes with lysosomes was observed when compared with their CS/DNA counterparts. A greater cellular uptake together with a less entry into lysosomes might thus explain the promotion of transfection efficiency of CS/DNA/gamma-PGA complexes. Knowledge of these mechanisms involving CS-based complexes containing gamma-PGA is critical for the development of an efficient vector for DNA transfection. (C) 2010 Elsevier Ltd. All rights reserved.