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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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Enhanced cellular retention and efficacy of siAF647-LNPs in NPC1 deficient MEFsa. Immunoblot analysis with anti-NPC1 antibody was used to validate wild type and NPC1 deficient MEFs. b. Automated confocal microscopy on NPC1+/+ or NPC1−/− MEFs exposed to different concentrations of labeled LNPs and imaged 24 hrs post incubation. Inset from a representative image (100 nM) shows siRNA accumulation in individual cells. c. Flow cytometery analysis on LNP-siRNA uptake in (i) NPC1+/+ or NPC1−/− cells or in (ii) NPC1−/− cells transfected with pEGFP or pNPC1-GFP. The mean fluorescent intensity represents LNP uptake. d. NPC1−/− cells treated with LNP-AF647-siRNA (red) (3 hrs pulse, 30 min chase) and immuno-stained with anti-LBPA antibody (green) e. LNPs containing siRNA against β integrin were added to wild type or NPC1 deficient MEFs as in (a), mRNA levels were quantitated at 24 hrs post incubation using branched DNA analysis. The experiment was done in triplicate and the errors are reported as S.E.M. f. A schematic representation of LNP trafficking (i) in NPC1+/+ and (ii) NPC1−/− cells. Intact cationic LNPs enter through macropinocytosis (1); a small fraction of LNPs transport from macropinosomes to the endocytic recycling compartment (ERC) (2) while the majority is directed to late endosomes (3). Late endosome sort LNPs to lysosomes for degradation or utilize multiple recycling pathways to traffic them to the extracellular milieu. These mechanisms include recycling through transport to the ER-Golgi route (4) or direct fusion of late endosomes containing multivesicular bodies, with the plasma membrane (Exosomes secretion) (5). In NPC1 deficient cells the late endosome recycling mechanisms are impaired causing LNP-siRNA to accumulate in enlarged late endosomes leading to persistent escape of siRNA that improves gene silencing. (Intact nanoparticle-, siRNA complex-)
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Figure 4: Enhanced cellular retention and efficacy of siAF647-LNPs in NPC1 deficient MEFsa. Immunoblot analysis with anti-NPC1 antibody was used to validate wild type and NPC1 deficient MEFs. b. Automated confocal microscopy on NPC1+/+ or NPC1−/− MEFs exposed to different concentrations of labeled LNPs and imaged 24 hrs post incubation. Inset from a representative image (100 nM) shows siRNA accumulation in individual cells. c. Flow cytometery analysis on LNP-siRNA uptake in (i) NPC1+/+ or NPC1−/− cells or in (ii) NPC1−/− cells transfected with pEGFP or pNPC1-GFP. The mean fluorescent intensity represents LNP uptake. d. NPC1−/− cells treated with LNP-AF647-siRNA (red) (3 hrs pulse, 30 min chase) and immuno-stained with anti-LBPA antibody (green) e. LNPs containing siRNA against β integrin were added to wild type or NPC1 deficient MEFs as in (a), mRNA levels were quantitated at 24 hrs post incubation using branched DNA analysis. The experiment was done in triplicate and the errors are reported as S.E.M. f. A schematic representation of LNP trafficking (i) in NPC1+/+ and (ii) NPC1−/− cells. Intact cationic LNPs enter through macropinocytosis (1); a small fraction of LNPs transport from macropinosomes to the endocytic recycling compartment (ERC) (2) while the majority is directed to late endosomes (3). Late endosome sort LNPs to lysosomes for degradation or utilize multiple recycling pathways to traffic them to the extracellular milieu. These mechanisms include recycling through transport to the ER-Golgi route (4) or direct fusion of late endosomes containing multivesicular bodies, with the plasma membrane (Exosomes secretion) (5). In NPC1 deficient cells the late endosome recycling mechanisms are impaired causing LNP-siRNA to accumulate in enlarged late endosomes leading to persistent escape of siRNA that improves gene silencing. (Intact nanoparticle-, siRNA complex-)

Mentions: Subcellular trafficking of lipids (e.g. cholesterol) from late endosomes/lysosomes towards the extracellular milieu has been reported to utilize the thirteen transmembrane glycoprotein NPC1 which is located on the surface of multivesicular late endosomes. Absence of NPC1 causes late endo/lysosomal dysfunction, cholesterol accumulation and is implicated in a lysosomal storage disease that causes liver and neural degeneration in human patients and animal models27–29. As the components of an LNP show some similarities to the endogenous lipids that use NPC1 as a receptor for recycling, we compared LNP retention in mouse embryonic fibroblasts (MEFs) devoid of NPC1 (NPC1−/−) with their wild type counterparts (NPC1+/+ MEFs) (Fig 4a). NPC1 deficient cells accumulate cholesterol due to defects in recycling (Fig S6). A markedly increased level (approximately 15 fold) of AF647-siRNA was observed in perinuclear enlarged endosomes in NPC1−/− compared to NPC1+/+ MEFs at 24 hrs post incubation, presumably due to enhanced cellular retention at a wide range of LNP concentrations (Fig 4a-b). Furthermore, enhanced cellular retention was observed in multiple cell types deficient in NPC1 including those isolated from human patients (FigS7a-b). We further tested for differences in the kinetics of LNP uptake in these cells. NPC1−/− cells show increased accumulation of siRNA after 2 hour of incubation as compared to NPC1+/+ cells where at early time points the amount of internalization is not effected. Moreover, a rescue experiment where we transfected an NPC1-GFP plasmid into NPC1−/− cells reduced the amount of intracellular LNP-siRNA to that of wild type cells (4c (i-ii)). We conclude that the increased level of siRNA in late endosomes is based on accumulation of LNPs due to defects in constitutively active NPC1 mediated recycling. Small molecules that impair cholesterol metabolism28 and increase cholesterol accumulation in cells failed to yield a significant increase in LNP accumulation (Fig S7c). Thus, the increased accumulation of LNPs is not due to just increased endosomal cholesterol but rather due to lack of direct interaction with putative intraluminal domains of NPC1 required for egress of LNPs. NPC1−/− cells have been reported to have high numbers of enlarged late endosomes, containing intraluminal vesicles enriched in lysobisphosphatidic acid (LBPA)29. LNPs accumulate in these structures and showed co-localization with antibody against LBPA in these cells (Fig 4d).


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)

Enhanced cellular retention and efficacy of siAF647-LNPs in NPC1 deficient MEFsa. Immunoblot analysis with anti-NPC1 antibody was used to validate wild type and NPC1 deficient MEFs. b. Automated confocal microscopy on NPC1+/+ or NPC1−/− MEFs exposed to different concentrations of labeled LNPs and imaged 24 hrs post incubation. Inset from a representative image (100 nM) shows siRNA accumulation in individual cells. c. Flow cytometery analysis on LNP-siRNA uptake in (i) NPC1+/+ or NPC1−/− cells or in (ii) NPC1−/− cells transfected with pEGFP or pNPC1-GFP. The mean fluorescent intensity represents LNP uptake. d. NPC1−/− cells treated with LNP-AF647-siRNA (red) (3 hrs pulse, 30 min chase) and immuno-stained with anti-LBPA antibody (green) e. LNPs containing siRNA against β integrin were added to wild type or NPC1 deficient MEFs as in (a), mRNA levels were quantitated at 24 hrs post incubation using branched DNA analysis. The experiment was done in triplicate and the errors are reported as S.E.M. f. A schematic representation of LNP trafficking (i) in NPC1+/+ and (ii) NPC1−/− cells. Intact cationic LNPs enter through macropinocytosis (1); a small fraction of LNPs transport from macropinosomes to the endocytic recycling compartment (ERC) (2) while the majority is directed to late endosomes (3). Late endosome sort LNPs to lysosomes for degradation or utilize multiple recycling pathways to traffic them to the extracellular milieu. These mechanisms include recycling through transport to the ER-Golgi route (4) or direct fusion of late endosomes containing multivesicular bodies, with the plasma membrane (Exosomes secretion) (5). In NPC1 deficient cells the late endosome recycling mechanisms are impaired causing LNP-siRNA to accumulate in enlarged late endosomes leading to persistent escape of siRNA that improves gene silencing. (Intact nanoparticle-, siRNA complex-)
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Related In: Results  -  Collection

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Figure 4: Enhanced cellular retention and efficacy of siAF647-LNPs in NPC1 deficient MEFsa. Immunoblot analysis with anti-NPC1 antibody was used to validate wild type and NPC1 deficient MEFs. b. Automated confocal microscopy on NPC1+/+ or NPC1−/− MEFs exposed to different concentrations of labeled LNPs and imaged 24 hrs post incubation. Inset from a representative image (100 nM) shows siRNA accumulation in individual cells. c. Flow cytometery analysis on LNP-siRNA uptake in (i) NPC1+/+ or NPC1−/− cells or in (ii) NPC1−/− cells transfected with pEGFP or pNPC1-GFP. The mean fluorescent intensity represents LNP uptake. d. NPC1−/− cells treated with LNP-AF647-siRNA (red) (3 hrs pulse, 30 min chase) and immuno-stained with anti-LBPA antibody (green) e. LNPs containing siRNA against β integrin were added to wild type or NPC1 deficient MEFs as in (a), mRNA levels were quantitated at 24 hrs post incubation using branched DNA analysis. The experiment was done in triplicate and the errors are reported as S.E.M. f. A schematic representation of LNP trafficking (i) in NPC1+/+ and (ii) NPC1−/− cells. Intact cationic LNPs enter through macropinocytosis (1); a small fraction of LNPs transport from macropinosomes to the endocytic recycling compartment (ERC) (2) while the majority is directed to late endosomes (3). Late endosome sort LNPs to lysosomes for degradation or utilize multiple recycling pathways to traffic them to the extracellular milieu. These mechanisms include recycling through transport to the ER-Golgi route (4) or direct fusion of late endosomes containing multivesicular bodies, with the plasma membrane (Exosomes secretion) (5). In NPC1 deficient cells the late endosome recycling mechanisms are impaired causing LNP-siRNA to accumulate in enlarged late endosomes leading to persistent escape of siRNA that improves gene silencing. (Intact nanoparticle-, siRNA complex-)
Mentions: Subcellular trafficking of lipids (e.g. cholesterol) from late endosomes/lysosomes towards the extracellular milieu has been reported to utilize the thirteen transmembrane glycoprotein NPC1 which is located on the surface of multivesicular late endosomes. Absence of NPC1 causes late endo/lysosomal dysfunction, cholesterol accumulation and is implicated in a lysosomal storage disease that causes liver and neural degeneration in human patients and animal models27–29. As the components of an LNP show some similarities to the endogenous lipids that use NPC1 as a receptor for recycling, we compared LNP retention in mouse embryonic fibroblasts (MEFs) devoid of NPC1 (NPC1−/−) with their wild type counterparts (NPC1+/+ MEFs) (Fig 4a). NPC1 deficient cells accumulate cholesterol due to defects in recycling (Fig S6). A markedly increased level (approximately 15 fold) of AF647-siRNA was observed in perinuclear enlarged endosomes in NPC1−/− compared to NPC1+/+ MEFs at 24 hrs post incubation, presumably due to enhanced cellular retention at a wide range of LNP concentrations (Fig 4a-b). Furthermore, enhanced cellular retention was observed in multiple cell types deficient in NPC1 including those isolated from human patients (FigS7a-b). We further tested for differences in the kinetics of LNP uptake in these cells. NPC1−/− cells show increased accumulation of siRNA after 2 hour of incubation as compared to NPC1+/+ cells where at early time points the amount of internalization is not effected. Moreover, a rescue experiment where we transfected an NPC1-GFP plasmid into NPC1−/− cells reduced the amount of intracellular LNP-siRNA to that of wild type cells (4c (i-ii)). We conclude that the increased level of siRNA in late endosomes is based on accumulation of LNPs due to defects in constitutively active NPC1 mediated recycling. Small molecules that impair cholesterol metabolism28 and increase cholesterol accumulation in cells failed to yield a significant increase in LNP accumulation (Fig S7c). Thus, the increased accumulation of LNPs is not due to just increased endosomal cholesterol but rather due to lack of direct interaction with putative intraluminal domains of NPC1 required for egress of LNPs. NPC1−/− cells have been reported to have high numbers of enlarged late endosomes, containing intraluminal vesicles enriched in lysobisphosphatidic acid (LBPA)29. LNPs accumulate in these structures and showed co-localization with antibody against LBPA in these cells (Fig 4d).

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