Limits...
Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling.

Sahay G, Querbes W, Alabi C, Eltoukhy A, Sarkar S, Zurenko C, Karagiannis E, Love K, Chen D, Zoncu R, Buganim Y, Schroeder A, Langer R, Anderson DG - Nat. Biotechnol. (2013)

Bottom Line: We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR and cathepsins. siRNA delivery is substantially reduced as ≅70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes.NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes and lysosomes, and increased gene silencing of the target gene.Our data suggest that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways.

View Article: PubMed Central - PubMed

Affiliation: The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

ABSTRACT
Despite efforts to understand the interactions between nanoparticles and cells, the cellular processes that determine the efficiency of intracellular drug delivery remain unclear. Here we examine cellular uptake of short interfering RNA (siRNA) delivered in lipid nanoparticles (LNPs) using cellular trafficking probes in combination with automated high-throughput confocal microscopy. We also employed defined perturbations of cellular pathways paired with systems biology approaches to uncover protein-protein and protein-small molecule interactions. We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR and cathepsins. siRNA delivery is substantially reduced as ≅70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes. Niemann-Pick type C1 (NPC1) is shown to be an important regulator of the major recycling pathways of LNP-delivered siRNAs. NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes and lysosomes, and increased gene silencing of the target gene. Our data suggest that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways.

Show MeSH

Related in: MedlinePlus

Cellular trafficking of LNPsa. Quantitative image analysis of siAF647-LNP cellular uptake (3 hrs) in HeLa cells silenced with siRNA against key endocytic regulators (Cdc42, Rac-1, Clathrin heavy chain (CHC), caveolin-1 (Cav-1). siRNA against luciferase serve as a negative control. (b-c) Image based quantitative analysis of siAF647-LNPs co-localization (3 hrs pulse, 15, 30, 60 min. chase) with markers of endocytosis, anti-EEA-1 (early endosomes), anti-LAMP-1 (late endosome/lysosomes), anti-LAMP-2 (late endosomes/lysosomes), Rab11-GFP and anti-Rab11 (both mark endocytic recycling compartment). High-resolution z-stack confocal images representative of 60 min chase are presented in different panels (b). Quantification of LNP-siRNA cellular uptake after indicated times (a, c).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3814166&req=5

Figure 2: Cellular trafficking of LNPsa. Quantitative image analysis of siAF647-LNP cellular uptake (3 hrs) in HeLa cells silenced with siRNA against key endocytic regulators (Cdc42, Rac-1, Clathrin heavy chain (CHC), caveolin-1 (Cav-1). siRNA against luciferase serve as a negative control. (b-c) Image based quantitative analysis of siAF647-LNPs co-localization (3 hrs pulse, 15, 30, 60 min. chase) with markers of endocytosis, anti-EEA-1 (early endosomes), anti-LAMP-1 (late endosome/lysosomes), anti-LAMP-2 (late endosomes/lysosomes), Rab11-GFP and anti-Rab11 (both mark endocytic recycling compartment). High-resolution z-stack confocal images representative of 60 min chase are presented in different panels (b). Quantification of LNP-siRNA cellular uptake after indicated times (a, c).

Mentions: To identify internalization pathways that are required for cationic LNP entry into cells we depleted key endocytic regulators using siRNA in HeLa cells. Down-regulation of Cdc42 and Rac1 (regulators of macropinocytosis) led to ca. 80% decrease in LNP uptake whereas inhibition of clathrin heavy chain-1 and caveolin-1 (regulators of clathrin and caveolae mediate endocytosis, respectively) had little impact on LNP entry (Fig 2a). Further transport of LNPs to select endocytic compartments in these cells was analyzed through co-localization studies based on endocytic markers. First, LNP initial entry through macropinocytosis was confirmed through strong localization with Cdc42-GFP and ovalbumin positive vesicles (both markers for macropinocytosis) as compared to that with Arf6-GFP positive vesicles (clathrin-and dynamin-independent pathways) (Fig S3a). Second, image based kinetics of LNP delivery to the general endo/lysosomal system revealed little co-localization of LNPs with EEA-1 or Rab5-RFP (early endosome markers) (post 3 hr incubation, at multiple time chase points, ca. 5-10% co-localization) but a steady increase in co-localization with LAMP-1, LAMP-2, Rab7-GFP and Lysotracker positive vesicles (late endosome/lysosome marker) starting as early as after 15 min chase and showing a steady increase to around 50% localization with late endosomes/lysosomes after 60 min (Fig 2b-c, Fig S3b-c). Notably, after 60 min a fraction of the labeled siRNA starts to show co-localization with markers of the tubulovesicular endocytic recycling compartment (ERC) (Rab11-GFP, antibody against Rab-11, transferrin) whereas a decrease in localization with lysosomal positive vesicles was observed (Fig 2b-c, Fig S3c). Inhibition of Rab11 (Rab11 siRNA and dominant negative (DN)-Rab11) to interfere with early endocytic recycling causes a 1.5 fold reduction in LNP internalization (Fig S3d-e). This decreased internalization may be a result of reduced endocytic recycling of trafficking regulators required for LNP entry.


Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling.

Sahay G, Querbes W, Alabi C, Eltoukhy A, Sarkar S, Zurenko C, Karagiannis E, Love K, Chen D, Zoncu R, Buganim Y, Schroeder A, Langer R, Anderson DG - Nat. Biotechnol. (2013)

Cellular trafficking of LNPsa. Quantitative image analysis of siAF647-LNP cellular uptake (3 hrs) in HeLa cells silenced with siRNA against key endocytic regulators (Cdc42, Rac-1, Clathrin heavy chain (CHC), caveolin-1 (Cav-1). siRNA against luciferase serve as a negative control. (b-c) Image based quantitative analysis of siAF647-LNPs co-localization (3 hrs pulse, 15, 30, 60 min. chase) with markers of endocytosis, anti-EEA-1 (early endosomes), anti-LAMP-1 (late endosome/lysosomes), anti-LAMP-2 (late endosomes/lysosomes), Rab11-GFP and anti-Rab11 (both mark endocytic recycling compartment). High-resolution z-stack confocal images representative of 60 min chase are presented in different panels (b). Quantification of LNP-siRNA cellular uptake after indicated times (a, c).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814166&req=5

Figure 2: Cellular trafficking of LNPsa. Quantitative image analysis of siAF647-LNP cellular uptake (3 hrs) in HeLa cells silenced with siRNA against key endocytic regulators (Cdc42, Rac-1, Clathrin heavy chain (CHC), caveolin-1 (Cav-1). siRNA against luciferase serve as a negative control. (b-c) Image based quantitative analysis of siAF647-LNPs co-localization (3 hrs pulse, 15, 30, 60 min. chase) with markers of endocytosis, anti-EEA-1 (early endosomes), anti-LAMP-1 (late endosome/lysosomes), anti-LAMP-2 (late endosomes/lysosomes), Rab11-GFP and anti-Rab11 (both mark endocytic recycling compartment). High-resolution z-stack confocal images representative of 60 min chase are presented in different panels (b). Quantification of LNP-siRNA cellular uptake after indicated times (a, c).
Mentions: To identify internalization pathways that are required for cationic LNP entry into cells we depleted key endocytic regulators using siRNA in HeLa cells. Down-regulation of Cdc42 and Rac1 (regulators of macropinocytosis) led to ca. 80% decrease in LNP uptake whereas inhibition of clathrin heavy chain-1 and caveolin-1 (regulators of clathrin and caveolae mediate endocytosis, respectively) had little impact on LNP entry (Fig 2a). Further transport of LNPs to select endocytic compartments in these cells was analyzed through co-localization studies based on endocytic markers. First, LNP initial entry through macropinocytosis was confirmed through strong localization with Cdc42-GFP and ovalbumin positive vesicles (both markers for macropinocytosis) as compared to that with Arf6-GFP positive vesicles (clathrin-and dynamin-independent pathways) (Fig S3a). Second, image based kinetics of LNP delivery to the general endo/lysosomal system revealed little co-localization of LNPs with EEA-1 or Rab5-RFP (early endosome markers) (post 3 hr incubation, at multiple time chase points, ca. 5-10% co-localization) but a steady increase in co-localization with LAMP-1, LAMP-2, Rab7-GFP and Lysotracker positive vesicles (late endosome/lysosome marker) starting as early as after 15 min chase and showing a steady increase to around 50% localization with late endosomes/lysosomes after 60 min (Fig 2b-c, Fig S3b-c). Notably, after 60 min a fraction of the labeled siRNA starts to show co-localization with markers of the tubulovesicular endocytic recycling compartment (ERC) (Rab11-GFP, antibody against Rab-11, transferrin) whereas a decrease in localization with lysosomal positive vesicles was observed (Fig 2b-c, Fig S3c). Inhibition of Rab11 (Rab11 siRNA and dominant negative (DN)-Rab11) to interfere with early endocytic recycling causes a 1.5 fold reduction in LNP internalization (Fig S3d-e). This decreased internalization may be a result of reduced endocytic recycling of trafficking regulators required for LNP entry.

Bottom Line: We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR and cathepsins. siRNA delivery is substantially reduced as ≅70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes.NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes and lysosomes, and increased gene silencing of the target gene.Our data suggest that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways.

View Article: PubMed Central - PubMed

Affiliation: The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

ABSTRACT
Despite efforts to understand the interactions between nanoparticles and cells, the cellular processes that determine the efficiency of intracellular drug delivery remain unclear. Here we examine cellular uptake of short interfering RNA (siRNA) delivered in lipid nanoparticles (LNPs) using cellular trafficking probes in combination with automated high-throughput confocal microscopy. We also employed defined perturbations of cellular pathways paired with systems biology approaches to uncover protein-protein and protein-small molecule interactions. We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR and cathepsins. siRNA delivery is substantially reduced as ≅70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes. Niemann-Pick type C1 (NPC1) is shown to be an important regulator of the major recycling pathways of LNP-delivered siRNAs. NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes and lysosomes, and increased gene silencing of the target gene. Our data suggest that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways.

Show MeSH
Related in: MedlinePlus