Endocytosis Controls siRNA Efficiency: Implications for siRNA Delivery Vehicle Design and Cell-Specific Targeting

Abstract

While small interfering RNAs (siRNAs) are commonly used for laboratory studies, development of siRNA therapeutics has been slower than expected, due, in part, to a still limited understanding of the endocytosis and intracellular trafficking of siRNA-containing complexes. With the recent characterization of multiple clathrin-/ caveolin-independent endocytic pathways, that is, those mediated by Graf1, Arf6, and flotillin, it has become clear that the endocytic mechanism influences subsequent intracellular processing of the internalized cargo. To explore siRNA delivery in light of these findings, we developed a novel assay that differentiates uptake by each of the endocytic pathways and can be used to determine whether endocytosis by a pathway leads to the initiation of RNA interference (RNAi). Using Lipofectamine 2000 (LF2K), we determined the endocytosis pathway leading to active silencing (whether by clathrin, caveolin, Arf6, Graf1, flotillin, or macropinocytosis) across multiple cell types (HeLa, H1299, HEK293, and HepG2). We showed that LF2K is internalized by Graf1-, Arf6-, or flotillin-mediated endocytosis for the initiation of RNAi, depending on cell type. In addition, we found that a portion of siRNA-containing complexes is internalized by pathways that do not lead to initiation of silencing. Inhibition of these pathways enhanced intracellular levels of siRNAs with concomitant enhancement of silencing.

Barriers and Strategies of Cationic Liposomes for Cancer Gene Therapy

“… 108 Lysosomal activity, a wide range of biological process involved in digestive function and host defense, relates to the degradation of substances from endocytosis or autophagy. 109 Cell type 110 , 111 and physicochemical properties of CLs, including size, charge, lipid composition, surface modification, are important factors in endocytosis. 56 , 101 When considering the effect of particle characteristics on endocytosis, the most striking factor is usually the particle size, as clathrin-coated pit and caveolae are size-limited invaginations.…”

Targeting the Inside of Cells with Biologicals: Chemicals as a Delivery Strategy

“…Only part of the siRNA cargo of a lipid-based carrier was released from a vesicle to the cytosol [ 166 ], which was argued to be inconsistent with fusion, which was expected to result in complete cargo release [ 153 ]. Although lipoplexes were assumed to potentially fuse directly with the membrane by some [ 167 – 169 ], others reported the uptake to be energy dependent, and lipoplexes were concluded not to fuse with the plasma membrane, but to be endocytosed [ 169 , 170 ].…”

“…A comparison of uptake into four different cell lines showed internalization to occur through multiple endocytic pathways, but mostly only one endocytic pathway resulted in cargo delivery. Which pathway was productive was cell-type dependent [ 169 ]. Besides depending on the carrier, uptake routes are generally highly cell-type dependent [ 33 , 238 ].…”

“…Besides depending on the carrier, uptake routes are generally highly cell-type dependent [ 33 , 238 ]. In HEK293 cells, RNA interference (RNAi) was facilitated via Graf1-mediated endocytosis (GME) [ 169 ], a pathway via which also dextran is internalized [ 239 ]. Arf6-dependent endocytosis (ADE) initiated RNAi in HeLa and HepG2 cells [ 169 ].…”

“…In HEK293 cells, RNA interference (RNAi) was facilitated via Graf1-mediated endocytosis (GME) [ 169 ], a pathway via which also dextran is internalized [ 239 ]. Arf6-dependent endocytosis (ADE) initiated RNAi in HeLa and HepG2 cells [ 169 ]. Blocking “non-productive pathways”, i.e.…”