title: Modulating polyplex-mediated gene transfection by small-molecule regulators of autophagy.
abstract: Nonviral gene transfection mediated by cationic polymer/DNA polyplexes often imposes stress and toxicity to cells. To better understand the relationship between cellular stress responses and polyplex-mediated transfection, polyplex-induced early autophagy in mouse fibroblasts was characterized and the impact of autophagy modulation on transgene expression evaluated. Transmission electron microscopy revealed the formation of double-membraned autophagosome in the cytoplasm of polyplex-transfected cells. Immunofluorescence staining and microscopy revealed intracellular LC3 punctation that was characteristic of early autophagy activation. Elevated expression of autophagosome-associated LC3 II protein was also detected by Western blot. When cells were treated with small-molecule modulators of autophagy, polyplex-mediated gene transfection efficiency was significantly affected. 3-Methyladenine (3-MA), an early autophagy inhibitor, reduced transfection efficiency, whereas rapamycin, an autophagy inducer, enhanced transgene expression. Importantly, the observed functional impact on gene transfection by autophagy modulation was decoupled from that of other modes of cellular stress response (apoptosis/necrosis). Treatment of cells by 3-MA or rapamycin did not affect the level of intracellular reactive oxygen species (ROS) but did decrease or increase, respectively, nuclear localization of polyplex-delivered plasmid DNA. These findings suggest new possibilities of enhancing polyplex-mediated gene delivery by codelivery of small-molecule regulators of autophagy.
authors: Department of Biomedical Engineering, University of Minnesota , 7-105 Hasselmo Hall, 312 Church Street SE, Minneapolis, Minnesota 55455, United States.
authors: Xiao
authors: X
authors: Zhong
authors: David
authors: D
authors: Panus
authors: Weihang
authors: W
authors: Ji
authors: Chun
authors: C
authors: Wang
mesh_terms: D021581:Active Transport, Cell Nucleus; D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001343:Autophagy / Q000187:drug effects / Q000235:genetics*; D001702:Biopharmaceutics; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D051379:Mice; D046529:Microscopy, Electron, Transmission; D041681:NIH 3T3 Cells; D010588:Phagosomes / Q000235:genetics / Q000648:ultrastructure; D011108:Polymers; D017382:Reactive Oxygen Species / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology; D014162:Transfection / Q000379:methods*
chemical_list: D011108:Polymers; D017382:Reactive Oxygen Species; C025946:3-methyladenine; D004247:DNA; D000225:Adenine; D020123:Sirolimus
keywords: autophagy; cytotoxicity; gene delivery; polyplex; transfection