Intracellular processing of poly(ethylene imine)/ribozyme complexes can be observed in living cells by using confocal laser scanning microscopy and inhibitor experiments
Intracellular processing of poly(ethylene imine)/ribozyme complexes can be observed in living cells by using confocal laser scanning microscopy and inhibitor experiments
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DOI：
10.1023/a:1014212630566
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发表时间：
2002-02-01
影响因子：
3.7
通讯作者：
Kissel, T
中科院分区：
文献类型：
--
作者：
Merdan, T;Kunath, K;Kissel, T
目的.聚（乙烯亚胺）/核酸复合物的亚细胞加工的关键步骤，特别是内体/溶酶体逃逸，可视化通过使用活细胞共聚焦激光扫描显微镜（CSLM），以深入了解其机制。活细胞共聚焦显微镜被用来检查随着时间的推移的聚（乙烯亚胺）/核酶和聚（L-赖氨酸）/核酶复合物的细胞内的命运，在存在和不存在巴弗洛霉素A1，内体/溶酶体酸化的选择性抑制剂。通过共聚焦显微镜用荧光亲酸染料鉴定复合物积累的隔室。为了确认显微镜数据，在类似的实验条件下测定荧光素酶报告基因的表达。聚（乙烯亚胺）/核酶复合物积累在酸性囊泡，最有可能是溶酶体。复合物的释放发生在突然的事件中，很可能是由于这些细胞器的破裂。释放后，聚（乙烯亚胺）和核酶遍布整个细胞，在此期间，细胞质和细胞核之间的分布略有差异是可见的。没有观察到溶酶体逃逸与聚（L-赖氨酸）/核酶复合物或聚（乙烯亚胺）/核酶复合物与巴夫洛霉素A1一起应用时。聚乙烯亚胺/质粒复合物表现出较高的荧光素酶表达，当用巴弗洛霉素A1抑制溶酶体酸化时，荧光素酶表达降低约200倍。我们的数据提供，第一次，直接的实验证据的逃逸聚（乙烯亚胺）/核酸复合物的内体/溶酶体室。CLSM，结合活细胞显微镜，是一个很有前途的工具，用于研究在核酸/基因传递的复合物的亚细胞命运。
Purpose. Critical steps in the subcellular processing of poly(ethylene imine)/nucleic acid complexes, especially endosomal/lysosomal escape, were visualized by using living cell confocal laser scanning microscopy (CSLM) to obtain an insight into their mechanism.Methods. Living cell confocal microscopy was used to examine the intracellular fate of poly(ethylene imine)/ribozyme and poly(L-lysine)/ribozyme complexes over time, in the presence of and without bafilomycin A1, a selective inhibitor of endosomal/lysosomal acidification. The compartment of complex accumulation was identified by confocal microscopy with a fluorescent acidotropic dye. To confirm microscopic data, luciferase reporter gene expression was determined under similar experimental conditions.Results. Poly(ethylene imine)/ribozyme complexes accumulate in acidic vesicles, most probably lysosomes. Release of complexes occurs in a sudden event, very likely due to bursting of these organelles. After release, poly(ethylene imine) and ribozyme spread throughout the cell, during which slight differences in distribution between cytosol and nucleus are visible. No lysosomal escape was observed with poly (L-lysine)/ribozyme complexes or when poly(ethylene imine)/ribozyme complexes were applied together with bafilomycin A1. Poly(ethylene imine)/plasmid complexes exhibited a high luciferase expression, which was reduced approximately 200-fold when lysosomal acidification was suppressed with bafilomycin A1.Conclusions. Our data provide, for the first time, direct experimental evidence for the escape of poly(ethylene imine)/nucleic acid complexes from the endosomal/lysosomal compartment. CLSM, in conjunction with living cell microscopy, is a promising tool for studying the subcellular fate of polyplexes in nucleic acid/gene delivery.