Comprehensive Pneumology Center, Institute of Lung Biology and Disease, Helmholtz Zentrum Munchen, and Institute of Experimental Genetics, Helmholtz Zentrum Munchen, Germany.
Mol Pharm. 2010 Jun 7;7(3):727-37. doi: 10.1021/mp900278x.
Polyethylene imine (PEI) based polycations, successfully used for gene therapy or RNA interference in vitro as well as in vivo, have been shown to cause well-known adverse side effects, especially high cytotoxicity. Therefore, various modifications have been developed to improve safety and efficiency of these nonviral vector systems, but profound knowledge about the underlying mechanisms responsible for the high cytotoxicity of PEI is still missing. In this in vitro study, we focused on stress and toxicity pathways triggered by PEI-based vector systems to be used for pulmonary application and two well-known lung toxic particles: fine crystalline silica (CS) and nanosized ZnO (NZO). The cytotoxicity profiles of all stressors were investigated in alveolar epithelial-like type II cells (LA4) to define concentrations with matching toxicity levels (cell viability >60% and LDH release <10%) for subsequent qRT-PCR-based gene array analysis. Within the first 6 h pathway analysis revealed for CS an extrinsic apoptotic signaling (TNF pathway) in contrast to the intrinsic apoptotic pathway (mitochondrial signaling) which was induced by PEI 25 kDa after 24 h treatment. The following causative chain of events seems conceivable: reactive oxygen species derived from particle surface toxicity triggers TNF signaling in the case of CS, whereby endosomal swelling and rupture upon endocytotic PEI 25 kDa uptake causes intracellular stress and mitochondrial alterations, finally leading to apoptotic cell death at higher doses. PEG modification most notably reduced the cytotoxicity of PEI 25 kDa but increased proinflammatory signaling on mRNA and even protein level. Hence in view of the lung as a sensitive target organ this inflammatory stimulation might cause unwanted side effects related to respiratory and cardiovascular disorders. Thus further optimization of the PEI-based vector systems is still needed for pulmonary application.
聚乙烯亚胺（PEI）基聚阳离子，已成功用于体外和体内的基因治疗或 RNA 干扰，已被证明会引起众所周知的不良副作用，尤其是高细胞毒性。因此，已经开发了各种修饰方法来提高这些非病毒载体系统的安全性和效率，但对于导致 PEI 高细胞毒性的潜在机制仍缺乏深入了解。在这项体外研究中，我们专注于 PEI 基载体系统引发的应激和毒性途径，这些途径将用于肺部应用以及两种众所周知的肺毒性颗粒：细晶态二氧化硅（CS）和纳米氧化锌（NZO）。在肺泡上皮样 II 型细胞（LA4）中研究了所有应激源的细胞毒性谱，以确定具有匹配毒性水平（细胞活力>60%和 LDH 释放<10%）的浓度，以便随后进行基于 qRT-PCR 的基因芯片分析。在最初的 6 小时内，通路分析显示 CS 诱导了外在凋亡信号（TNF 途径），而与 PEI 25 kDa 24 小时处理后诱导的内在凋亡途径（线粒体信号）相反。以下因果链似乎是合理的：源自颗粒表面毒性的活性氧物种在 CS 的情况下触发 TNF 信号，而内吞的 PEI 25 kDa 摄取导致内体肿胀和破裂，从而导致细胞内应激和线粒体改变，最终在较高剂量下导致细胞凋亡。PEG 修饰最显著地降低了 PEI 25 kDa 的细胞毒性，但增加了 mRNA 甚至蛋白质水平的促炎信号。因此，鉴于肺作为敏感靶器官，这种炎症刺激可能会导致与呼吸和心血管疾病相关的不良副作用。因此，对于肺部应用，仍需要进一步优化 PEI 基载体系统。
