The ER is part of the endomembrane system, linking it to the Golgi apparatus, cell membrane, and lysosomes via trafficking of membrane encapsulated vesicles. Endocytosis of siRNA-containing complexes begins when an initiation event causes the cell membrane to envelop the complex forming an endosome. These vesicles are then trafficked to the sorting endosome that determines to which endomembrane organelle the cargo will be distributed. To achieve a therapeutic effect, siRNAs must escape the endomembrane system during vesicle processing by either delivery vehicle fusion with the vesicle membrane or forming pores in the vesicle membrane.
One change to the endomembrane system that occurs during ER stress is an increase in autophagy. Stress causing agents near the ER initiate a membrane formation event that forms a phagophore, which engulfs the problematic compounds. Phagophores mature into autophagosomes before merging with lysosomes for cargo degradation. Autophagy can remove material from the endomembrane system by fusing membrane bound vesicles with phagophores to sequester new material that is causing further demand on the ER during stress.
In this work, we sought to examine how autophagy associated with ER stress influenced endocytosis, endosomal escape, and function of siRNAs delivered by cationic lipids. HeLa cells constitutively expressing EGFP were treated with increasing levels of tunicamycin (TM), a common ER stress inducer, followed by transfection with Lipofectamine 2000/siRNA complexes targeting EGFP. TM treated cells accumulated higher levels of siRNA but had lower siRNA-induced silencing. 3-methyladenine was used to inhibit the effect of autophagy during TM treatment, which alleviated siRNA accumulation levels. To determine at what stage of endosome maturation siRNAs were being sequestered, we treated cells with Bafilomycin A1 (BAFA1) to prevent the acidification of early endosomes before becoming late endosomes or lysosomes. BAFA1 treatment resulted in a similar but diminished increase in siRNA accumulation and reduction in silencing, indicating that in both cases, siRNAs do not likely progress from early endosomes. Finally, to investigate if the ER stress-sequestered siRNAs can initiate silencing, we allowed cells to recover in TM and control media for 5 days after transfection. siRNA accumulation was increased and persisted longer with delayed maximal silencing. Taken together our results inform clinical studies of siRNA therapeutics, suggesting that conditions and comorbidities that cause increased autophagy may alter the endosome processing and cellular pharmacokinetic properties of cationic-lipid delivered siRNAs.