Biochim Biophys Acta Gen Subj . 2025 Nov 1;1870(1):130878.
Elevated cyclic hydrostatic pressure enhances the transfection activity of lipoplexes by activating clathrin-mediated endocytosis
Weichen Zhan 1, Xiaowei Ding 2, Zhongrui Cui 1, Yizhuo Wu 1, Yiwen Gu 1, Hanxiao Cheng 1, Xinxin Ge 3, Yun Wang 3, Jiangyun Luo 4, Bing Xiao 5
PMID: 41183625 DOI: 10.1016/j.bbagen.2025.130878
Abstract Despite significant advancements in liposome-mediated transfection technology over the past decades, achieving optimal transfection efficiency with lipoplex remains challenging in certain primary cells, such as vascular smooth muscle cells, endothelial cells, and suspension cells. Here, we present an innovative approach to significantly enhance Lipofectamine-based transient transfection efficiency in hard-to-transfect cells by applying elevated cyclic hydrostatic pressure (CHP). The plasmids encoding the enhanced green fluorescent protein (EGFP) were transfected using Lipofectamine 3000 reagent, and the transfection efficiency was evaluated by Western blot or flow cytometry. Our results demonstrate that CHP (0.0083 Hz, 0-100 mmHg) significantly enhanced the transfection efficiency of lipoplex in primary human aortic smooth muscle cells (HASMCs) and other difficult-to-transfect cell types. Mechanistic studies revealed that the enhancement of liposome-mediated transfection by CHP was dependent on the activation of clathrin-dependent endocytic pathways. Importantly, this mechanical stimulation did not affect the proliferative or migratory capacities of HASMCs, despite the identification of significantly modulated proteins (5.8 % of the total proteome) by proteomic analysis. This study establishes a novel, safe strategy to enhance lipoplex-mediated nucleic acid delivery in challenging-to-transfect cell types.
摘要中文翻译（AI）
摘要
尽管脂质体介导的转染技术在过去的几十年里取得了显著进展，但在某些原代细胞（如血管平滑肌细胞、内皮细胞和悬浮细胞）中，要实现脂质复合物（lipoplex）的最佳转染效率仍然具有挑战性。本文提出了一种创新方法，通过施加升高的循环静水压（CHP），来显著提高基于Lipofectamine的难转染细胞中的瞬时转染效率。研究使用Lipofectamine 3000试剂转染编码增强型绿色荧光蛋白（EGFP）的质粒，并通过蛋白质印迹法或流式细胞术评估转染效率。我们的结果表明，CHP（0.0083 Hz, 0-100 mmHg）能显著提高脂质复合物在原代人主动脉平滑肌细胞（HASMCs）及其他难转染细胞类型中的转染效率。机制研究表明，CHP对脂质体介导的转染的增强作用，依赖于网格蛋白依赖性内吞途径的激活。重要的是，尽管蛋白质组学分析鉴定出相当数量的蛋白质（占总蛋白质组的5.8%）发生了显著调控，但这种机械刺激并未影响HASMCs的增殖或迁移能力。本研究确立了一种新颖、安全的策略，可用于增强脂质复合物在难转染细胞类型中的核酸递送效率。
