Abstract：
Polymer-cationic mediated gene delivery is a well-stablished strategy of transient gene expression (TGE) in mammalian cell cultures. Nonetheless, its industrial implementation is hindered by the phenomenon known as cell density effect (CDE) that limits the cell density at which cultures can be efficiently transfected. The rise in personalized medicine and multiple cell and gene therapy approaches based on TGE, make more relevant to understand how to circumvent the CDE. A rational study upon DNA/PEI complex formation, stability and delivery during transfection of HEK293 cell cultures has been conducted, providing insights on the mechanisms for polyplexes uptake at low cell density and disruption at high cell density. DNA/PEI polyplexes were physiochemically characterized by coupling X-ray spectroscopy, confocal microscopy, cryo-transmission electron microscopy (TEM) and nuclear magnetic resonance (NMR). Our results showed that the ionic strength of polyplexes significantly increased upon their addition to exhausted media. This was reverted by depleting extracellular vesicles (EVs) from the media. The increase in ionic strength led to polyplex aggregation and prevented efficient cell transfection which could be counterbalanced by implementing a simple media replacement (MR) step before transfection. Inhibiting and labeling specific cell-surface proteoglycans (PGs) species revealed different roles of PGs in polyplexes uptake. Importantly, the polyplexes uptake process seemed to be triggered by a coalescence phenomenon of HSPG like glypican-4 around polyplex entry points. Ultimately, this study provides new insights into PEI-based cell transfection methodologies, enabling to enhance transient transfection and mitigate the cell density effect (CDE).
摘要：
聚合物阳离子介导的基因传递是哺乳动物细胞培养物中瞬时基因表达（TGE）的良好策略。尽管如此，其工业实施受到被称为细胞密度效应(CDE)的现象的阻碍,该现象限制了培养物可以有效转染的细胞密度。基于TGE的个性化医疗和多种细胞和基因治疗方法的兴起，让更多的相关了解如何规避CDE。对DNA/PEI复合物形成的理性研究，在HEK293细胞培养物转染期间的稳定性和递送已经进行，提供有关低细胞密度下聚合复合物摄取和高细胞密度下破坏的机制的见解。DNA/PEI复合物通过耦合X射线光谱法进行物理化学表征，共聚焦显微镜，低温透射电子显微镜(TEM)和核磁共振(NMR)。我们的结果表明，将复合物添加到耗尽的介质中后，其离子强度显着增加。这通过从培养基中耗尽细胞外囊泡(EV)来恢复。离子强度的增加导致复合物聚集并阻止有效的细胞转染,这可以通过在转染之前实施简单的培养基替换(MR)步骤来抵消。抑制和标记特定的细胞表面蛋白聚糖（PG）物种揭示了PG在聚合复合物摄取中的不同作用。重要的是,聚合复合物的摄取过程似乎是由HSPG的聚结现象触发的，例如在聚合复合物进入点周围的磷脂酰肌醇蛋白聚糖-4。最终,这项研究为基于PEI的细胞转染方法提供了新的见解，能够增强瞬时转染和减轻细胞密度效应（CDE）。
