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.

A mechanistic investigation exploring the differential transfection efficiencies between the easy-to-transfect SK-BR3 and difficult-to-transfect CT26 cell lines

“…This ruffling, coupled with the blebbing and irregular morphologies observed in the confocal images of CT26 cells (Fig. 4), may suggest that CT26 cells are more sensitive to foreign materials than SK-BR3, and may be signs of nanomaterial induced cellular toxicity [20]. Lastly, the vesicles imaged in CT26 cells at the 24-h time point appear to contain both particle aggregates and cellular debris; this observation suggests that the cytoplasmic vesicles containing the AuPAMAM/DNA complexes may have degradative properties, or may be responsible for sequestering degraded material.…”

Intracellular Trafficking and Endosomal Release of Oligonucleotides: What We Know and What We Don’t

“…An interesting emerging issue for both LNP and polyplex delivery is the potential role of autophagy. Thus, reports have indicated that damage to endosomes caused by delivery agents can trigger the autophagic process both for LPs and for polyplexes [37,51,52]. However, it is not clear to what degree autophagosome formation affects the functionality of the delivered oligonucleotide, with some reports indicating a significant effect via modulation of autophagy [51] and others indicating the opposite [37].…”

Identifying key barriers in cationic polymer gene delivery to human T cells

“…Autophagy regulators 3-methyladenine (3-MA) and rapamycin were used in transfection studies as previously described. 27 Briefly, 3-MA was supplemented to Opti-MEM medium at 5 or 10 mM during the 4 hour transfection period. The complete media added after transfection did not contain 3-MA.…”