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.

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Systems survey for endocytosis of lipid nanoparticles (LNP)LNPs containing siAF647 were incubated with HeLa-GFP cells (50nM) for 3 hrs in the presence or absence of small molecules and imaged using an automated high throughput confocal microscope a. Small molecules that inhibit over 80% of internalization (with no more than 10%-15% loss of cell viability) and their network interactions are presented in the form of a systems diagram b. Localization of siAF647LNP in presence or absence of bafilomycin is represented as an individual image (arrows indicate peripheral localization) and a 3D z-stack. c. Dual HeLa cells were exposed to LNP (siLuc, 10nM) in the presence or absence of bafilomycin (1μM). Luciferase to Renilla levels was measured for silencing activity. Experiments were performed in triplicate; errors are plotted as standard error means (S.E.M.). (d-e). Time lapse Total internal reflection (TIRF) microscopy of cells exposed to siAF647-LNPs (50nM, 3hrs) in the presence or absence of bafilomycin was subjected to multiple particle tracking (MPT). A snapshot of vesicular tracks indicating the movement of LNPs in presence or absence of bafilomycin presented in (d). The ratio between the MPT parameters for untreated/bafilomycin treated cells was calculated to provide a quantitative measure of differences in LNP trafficking with or without bafilomycin (e). Errors of ratios are plotted through propagation of errors from division.
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Figure 1: Systems survey for endocytosis of lipid nanoparticles (LNP)LNPs containing siAF647 were incubated with HeLa-GFP cells (50nM) for 3 hrs in the presence or absence of small molecules and imaged using an automated high throughput confocal microscope a. Small molecules that inhibit over 80% of internalization (with no more than 10%-15% loss of cell viability) and their network interactions are presented in the form of a systems diagram b. Localization of siAF647LNP in presence or absence of bafilomycin is represented as an individual image (arrows indicate peripheral localization) and a 3D z-stack. c. Dual HeLa cells were exposed to LNP (siLuc, 10nM) in the presence or absence of bafilomycin (1μM). Luciferase to Renilla levels was measured for silencing activity. Experiments were performed in triplicate; errors are plotted as standard error means (S.E.M.). (d-e). Time lapse Total internal reflection (TIRF) microscopy of cells exposed to siAF647-LNPs (50nM, 3hrs) in the presence or absence of bafilomycin was subjected to multiple particle tracking (MPT). A snapshot of vesicular tracks indicating the movement of LNPs in presence or absence of bafilomycin presented in (d). The ratio between the MPT parameters for untreated/bafilomycin treated cells was calculated to provide a quantitative measure of differences in LNP trafficking with or without bafilomycin (e). Errors of ratios are plotted through propagation of errors from division.

Mentions: When HeLa-GFP cells were incubated with LNPs containing Alexa-647-labelled siRNA against GFP for 3 hrs, the particles appear to localize at the perinuclear region within 1 hour post exposure (Fig S1 a-b). Subcellular localization of LNP-siRNA in the presence of small molecule inhibitors under similar conditions was visualized and the amount of internalized siRNA was quantified. The small molecule library consisted of molecules that affect cell signaling, intracellular trafficking as well as had the ability to either induce or inhibit autophagy (for a list of the inhibitors used and the quantification of their effect on LNP uptake see Supplemental Information Small Molecule Library). We considered the subset of small molecules that had substantial inhibition on LNP uptake and using databases (See Supplementary Information Methods) identified the proteins with which the small molecules interact with and created protein-protein and protein-small molecule interaction networks. This systems analysis identified several endocytic regulators required for LNP entry which includes VoATPase, mTOR (mammalian target of rapamycin), cathepsins, Protein Kinase C, NFκB, calcium channels, chloride channels and arachidonic acid metabolic pathways (Fig 1a). Previous work has identified, among other processes, signaling cascades such as mTOR and cathepsins, as regulators of the cellular uptake of glucose and viruses by macropinocytosis18,19. We have summarized known effectors of cellular trafficking in Table S1. Because previous studies show that interference with acidification impacts release of nucleic acids from endosomal compartments20, our results further corroborated the effects of proton pump on endocytosis of LNPs. We found that the VoATPase inhibitor, bafilomycin inhibits LNP entry inside cells (Fig 1b) whereas having no effect on the LNPs stability (FigS2a). Multiple LNPs with different cationic lipid compositions containing siRNA or Hydrogenated soybean phosphatidylcholine (HSPC)-based liposomes containing small molecules showed marked decrease in uptake in the presence of two different proton pump inhibitors, concanamycin and bafilomycin (FigS2b-c)21 . Furthermore, treatment with bafilomycin led to a decrease in siRNA-induced target gene silencing (Fig 1c).


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)

Systems survey for endocytosis of lipid nanoparticles (LNP)LNPs containing siAF647 were incubated with HeLa-GFP cells (50nM) for 3 hrs in the presence or absence of small molecules and imaged using an automated high throughput confocal microscope a. Small molecules that inhibit over 80% of internalization (with no more than 10%-15% loss of cell viability) and their network interactions are presented in the form of a systems diagram b. Localization of siAF647LNP in presence or absence of bafilomycin is represented as an individual image (arrows indicate peripheral localization) and a 3D z-stack. c. Dual HeLa cells were exposed to LNP (siLuc, 10nM) in the presence or absence of bafilomycin (1μM). Luciferase to Renilla levels was measured for silencing activity. Experiments were performed in triplicate; errors are plotted as standard error means (S.E.M.). (d-e). Time lapse Total internal reflection (TIRF) microscopy of cells exposed to siAF647-LNPs (50nM, 3hrs) in the presence or absence of bafilomycin was subjected to multiple particle tracking (MPT). A snapshot of vesicular tracks indicating the movement of LNPs in presence or absence of bafilomycin presented in (d). The ratio between the MPT parameters for untreated/bafilomycin treated cells was calculated to provide a quantitative measure of differences in LNP trafficking with or without bafilomycin (e). Errors of ratios are plotted through propagation of errors from division.
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Related In: Results  -  Collection

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Figure 1: Systems survey for endocytosis of lipid nanoparticles (LNP)LNPs containing siAF647 were incubated with HeLa-GFP cells (50nM) for 3 hrs in the presence or absence of small molecules and imaged using an automated high throughput confocal microscope a. Small molecules that inhibit over 80% of internalization (with no more than 10%-15% loss of cell viability) and their network interactions are presented in the form of a systems diagram b. Localization of siAF647LNP in presence or absence of bafilomycin is represented as an individual image (arrows indicate peripheral localization) and a 3D z-stack. c. Dual HeLa cells were exposed to LNP (siLuc, 10nM) in the presence or absence of bafilomycin (1μM). Luciferase to Renilla levels was measured for silencing activity. Experiments were performed in triplicate; errors are plotted as standard error means (S.E.M.). (d-e). Time lapse Total internal reflection (TIRF) microscopy of cells exposed to siAF647-LNPs (50nM, 3hrs) in the presence or absence of bafilomycin was subjected to multiple particle tracking (MPT). A snapshot of vesicular tracks indicating the movement of LNPs in presence or absence of bafilomycin presented in (d). The ratio between the MPT parameters for untreated/bafilomycin treated cells was calculated to provide a quantitative measure of differences in LNP trafficking with or without bafilomycin (e). Errors of ratios are plotted through propagation of errors from division.
Mentions: When HeLa-GFP cells were incubated with LNPs containing Alexa-647-labelled siRNA against GFP for 3 hrs, the particles appear to localize at the perinuclear region within 1 hour post exposure (Fig S1 a-b). Subcellular localization of LNP-siRNA in the presence of small molecule inhibitors under similar conditions was visualized and the amount of internalized siRNA was quantified. The small molecule library consisted of molecules that affect cell signaling, intracellular trafficking as well as had the ability to either induce or inhibit autophagy (for a list of the inhibitors used and the quantification of their effect on LNP uptake see Supplemental Information Small Molecule Library). We considered the subset of small molecules that had substantial inhibition on LNP uptake and using databases (See Supplementary Information Methods) identified the proteins with which the small molecules interact with and created protein-protein and protein-small molecule interaction networks. This systems analysis identified several endocytic regulators required for LNP entry which includes VoATPase, mTOR (mammalian target of rapamycin), cathepsins, Protein Kinase C, NFκB, calcium channels, chloride channels and arachidonic acid metabolic pathways (Fig 1a). Previous work has identified, among other processes, signaling cascades such as mTOR and cathepsins, as regulators of the cellular uptake of glucose and viruses by macropinocytosis18,19. We have summarized known effectors of cellular trafficking in Table S1. Because previous studies show that interference with acidification impacts release of nucleic acids from endosomal compartments20, our results further corroborated the effects of proton pump on endocytosis of LNPs. We found that the VoATPase inhibitor, bafilomycin inhibits LNP entry inside cells (Fig 1b) whereas having no effect on the LNPs stability (FigS2a). Multiple LNPs with different cationic lipid compositions containing siRNA or Hydrogenated soybean phosphatidylcholine (HSPC)-based liposomes containing small molecules showed marked decrease in uptake in the presence of two different proton pump inhibitors, concanamycin and bafilomycin (FigS2b-c)21 . Furthermore, treatment with bafilomycin led to a decrease in siRNA-induced target gene silencing (Fig 1c).

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