Autophagy plays a dual role during intracellular siRNA delivery by lipoplex and polyplex nanoparticles

Abstract

Growing evidence indicates that autophagy plays a vital role during intracellular DNA delivery mediated by lipoplex and polyplex nanoparticles. However, autophagy in intracellular siRNA delivery has not been well understood. In this study, lipofectamine 2000 and chitosan were used to formulate lipoplex and polyplex with siRNA for systematically investigating the interplay between siRNA delivery and autophagy. After transfection of H1299 cells with lipoplex and polyplex, the number of autophagic vacuoles was increased significantly indicated by the accumulation of monodansylcadaverine (MDC) staining. Western blot revealed that the LC3-II expression was significantly increased after transfection, whereas p-mTOR expression was not influenced apparently. In addition, small-molecule autophagy modulators significantly affected transfection efficiency. Specifically, the mTOR-dependent autophagy inducer rapamycin enhanced the knockdown efficiency of both lipoplex and polyplex, whereas mTOR-dependent autophagy inhibitor 3-methyladenine (3-MA) suppressed their silencing efficiency. On the contrary, mTOR-independent autophagy inducer LiBr decreased whereas mTOR-independent autophagy inhibitor thapsigargin (TG) increased the knockdown efficacy. Immunofluorescence staining showed that siRNA was partially co-localized with autophagosomes and the percentage of co-localized siRNA was significantly affected by autophagy modulators in the opposite trend of gene knockdown efficacy. In conclusion, our study suggests that autophagy plays an important role during the intracellular siRNA trafficking mediated by both lipoplex and polyplex. Modulating autophagy process will result in distinct knockdown efficiency, which may be applied as a potential convenient way for improving siRNA delivery efficacy.

Statement of significance: Although tremendous effects has been made in the development of non-viral siRNA delivery systems, the intracellular siRNA trafficking has not been elucidated clearly. In this study, we systematically investigated the relationship between autophagy and intracellular siRNA delivery. We found that the non-viral siRNA delivery by both lipoplex and polyplex could induce mTOR-independent autophagy response. More interestingly, knockdown efficiency of both lipoplex and polyplex could be modulated with different autophagy regulators. Specifically, the mTOR-dependent autophagy inducer rapamycin enhances the knockdown efficiency of both lipoplex and polyplex, whereas mTOR-dependent autophagy inhibitor 3-methyladenine suppresses their silencing efficiency. On the contrary, mTOR-independent autophagy inducer lithium bromide decreases, whereas mTOR-independent autophagy inhibitor thapsigargin increases the knockdown efficacy. These findings suggest that the mTOR-dependent and -independent autophagy play a distinct role in the intracellular siRNA trafficking. Furthermore, co-administration with proper autophagy regulators could be potential convenient method to modulate siRNA transfection efficacy.

Keywords: Autophagy; Lipoplex; Nanoparticles; Polyplex; siRNA delivery.