School of Pharmacy, University of Eastern Finland, P.O. Box 1627, 70211 Kuopio, Finland; Department of Pharmaceutics, School of Pharmacy, Zanjan University of Medical Sciences, P.O. Box 56184, 45139 Zanjan, Iran.
School of Pharmacy, University of Eastern Finland, P.O. Box 1627, 70211 Kuopio, Finland.
J Control Release. 2014 Aug 10;187:59-65. doi: 10.1016/j.jconrel.2014.05.005. Epub 2014 May 14.
Intracellular limiting steps and molecules involved in internalization and intracellular routing of non-viral gene delivery systems are still poorly understood. In this study, the intracellular kinetics of three different gene delivery systems calcium phosphate precipitates (CaP), polyethyleneimine (PEI) and N-[1-(2,3-dioleyl)propyl]-N,N,N-trimethylammonium chloride (DOTAP)) were quantified at cellular, nuclear, transcriptional and translational levels by using qRT-PCR. Additionally, a role of cell surface glycosaminoglycans (GAGs) was evaluated by performing the aforementioned studies in cells devoid of GAGs (pgsB-618) and cells lacking heparan sulphate (HS). The obtained data showed that the intracellular kinetics was dependent on the type of gene carrier and the weakest intracellular step varied between the carriers; rapid elimination of cell-associated pDNA in CaP, nuclear uptake in DOTAP and transcriptional and translational events in PEI mediated transfections. Overall, neither the amount of cell- nor nuclear associated pDNA correlated with transgene expression but the mRNA expression of the transgene correlated well with the expression at protein level. The nuclear uptake of pDNA in all cases was rapid and efficient thus indicating that the post-nuclear processes including transcription and translation steps have a critical role in defining the efficiency of non-viral gene delivery systems. Our study demonstrated that cell-surface GAGs are not essential for cell surface binding and internalization of gene delivery complexes, but they are able to define the intracellular routing of the complexes by leading them to pathways with high pDNA elimination.
细胞内摄取和细胞内转运非病毒基因传递系统的限制步骤和涉及的分子仍未得到很好的理解。在这项研究中，通过 qRT-PCR 在细胞、核、转录和翻译水平上定量了三种不同基因传递系统（磷酸钙沉淀（CaP）、聚乙烯亚胺（PEI）和 N-[1-(2,3-二油酰基)丙基]-N,N,N-三甲基氯化铵（DOTAP））的细胞内动力学。此外，通过在缺乏糖胺聚糖（GAGs）的细胞（pgsB-618）和缺乏肝素硫酸盐（HS）的细胞中进行上述研究，评估了细胞表面 GAGs 的作用。获得的数据表明，细胞内动力学取决于基因载体的类型，载体之间的最弱细胞内步骤也不同；CaP 中细胞相关 pDNA 的快速消除、DOTAP 中的核摄取以及 PEI 介导的转染中的转录和翻译事件。总体而言，细胞或核相关 pDNA 的量与转基因表达均无相关性，但转基因的 mRNA 表达与蛋白水平的表达相关性良好。在所有情况下，pDNA 的核摄取都迅速且高效，这表明包括转录和翻译步骤在内的核后过程在定义非病毒基因传递系统的效率方面起着关键作用。我们的研究表明，细胞表面 GAGs 对于基因传递复合物的细胞表面结合和内化不是必需的，但它们能够通过将复合物引导至具有高 pDNA 消除的途径来定义复合物的细胞内途径。
