Letter | Published:

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

Nature Biotechnology volume 31, pages 653658 (2013) | Download Citation


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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).

  2. 2.

    , & Knocking down barriers: advances in siRNA delivery. Nat. Rev. Drug Discov. 8, 129–138 (2009).

  3. 3.

    et al. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 464, 1067–1070 (2010).

  4. 4.

    Proof of concept for next-generation nanoparticle drugs in humans. Nat. Biotechnol. 30, 471–473 (2012).

  5. 5.

    , & Endocytosis of nanomedicines. J. Control. Release 145, 182–195 (2010).

  6. 6.

    , , & More effective nanomedicines through particle design. Small 7, 1919–1931 (2011).

  7. 7.

    , & Cellular uptake and intracellular trafficking of antisense and siRNA oligonucleotides. Bioconjug. Chem. 23, 147–157 (2012).

  8. 8.

    , & Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes. J. Biol. Chem. 278, 44826–44831 (2003).

  9. 9.

    et al. Influence of cationic lipid composition on gene silencing properties of lipid nanoparticle formulations of siRNA in antigen-presenting cells. Mol. Ther. 19, 2186–2200 (2011).

  10. 10.

    et al. Real-time gene delivery vector tracking in the endo-lysosomal pathway of live cells. Microsc. Res. Tech. 75, 691–697 (2012).

  11. 11.

    , & A novel mechanism is involved in cationic lipid-mediated functional siRNA delivery. Mol. Pharm. 6, 763–771 (2009).

  12. 12.

    & Nucleic acid delivery: the missing pieces of the puzzle? Acc. Chem. Res. 45, 1153–1162 (2012).

  13. 13.

    et al. Systems survey of endocytosis by multiparametric image analysis. Nature 464, 243–249 (2010).

  14. 14.

    & Mechanisms of endocytosis. Annu. Rev. Biochem. 78, 857–902 (2009).

  15. 15.

    et al. Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation. J. Am. Chem. Soc. 134, 6948–6951 (2012).

  16. 16.

    et al. Lipid-like materials for low-dose, in vivo gene silencing. Proc. Natl. Acad. Sci. USA 107, 1864–1869 (2010).

  17. 17.

    et al. Therapeutic siRNA silencing in inflammatory monocytes. Nat. Biotechnol. 29, 1005–1010 (2011).

  18. 18.

    et al. mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H(+)-ATPase. Science 334, 678–683 (2011).

  19. 19.

    et al. Ebola virus entry requires the cholesterol transporter Niemann-Pick C1. Nature 477, 340–343 (2011).

  20. 20.

    , , & Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery. Pharmacol. Rev. 58, 32–45 (2006).

  21. 21.

    et al. Concanamycin A, the specific inhibitor of V-ATPases, binds to the V(o) subunit c. J. Biol. Chem. 277, 40544–40548 (2002).

  22. 22.

    et al. Effect of bafilomycin A1 and nocodazole on endocytic transport in HeLa cells: implications for viral uncoating and infection. J. Virol. 72, 9645–9655 (1998).

  23. 23.

    et al. Regulation of mammalian autophagy in physiology and pathophysiology. Physiol. Rev. 90, 1383–1435 (2010).

  24. 24.

    et al. Complex inhibitory effects of nitric oxide on autophagy. Mol. Cell 43, 19–32 (2011).

  25. 25.

    et al. Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Mol. Ther. 18, 1357–1364 (2010).

  26. 26.

    et al. FRET-labeled siRNA probes for tracking assembly and disassembly of siRNA nanocomplexes. ACS Nano 6, 6133–6141 (2012).

  27. 27.

    Cellular cholesterol trafficking and compartmentalization. Nat. Rev. Mol. Cell Biol. 9, 125–138 (2008).

  28. 28.

    , , & Dynamic movements of organelles containing Niemann-Pick C1 protein: NPC1 involvement in late endocytic events. Mol. Biol. Cell 12, 601–614 (2001).

  29. 29.

    et al. Cessation of rapid late endosomal tubulovesicular trafficking in Niemann–Pick type C1 disease. Proc. Natl. Acad. Sci. USA 98, 4466–4471 (2001).

  30. 30.

    , , & Endocytic trafficking of sphingomyelin depends on its acyl chain length. Mol. Biol. Cell 18, 5113–5123 (2007).

  31. 31.

    , , & Characterization of the intracellular dynamics of a non-degradative pathway accessed by polymer nanoparticles. J. Control. Release 125, 107–111 (2008).

  32. 32.

    et al. Rab8-dependent recycling promotes endosomal cholesterol removal in normal and sphingolipidosis cells. Mol. Biol. Cell 18, 47–56 (2007).

  33. 33.

    & The Rab GTPase family. Genome Biol. 2, REVIEWSS3007 (2001).

  34. 34.

    et al. Rab27a and Rab27b control different steps of the exosome secretion pathway. Nat. Cell Biol. 12, 19–30 (2010).

  35. 35.

    , , , & Modulation of cellular cholesterol transport and homeostasis by Rab11. Mol. Biol. Cell 13, 3107–3122 (2002).

  36. 36.

    , , & Multivesicular bodies associate with components of miRNA effector complexes and modulate miRNA activity. Nat. Cell Biol. 11, 1143–1149 (2009).

  37. 37.

    et al. Silencing by small RNAs is linked to endosomal trafficking. Nat. Cell Biol. 11, 1150–1156 (2009).

  38. 38.

    & RISC hitches onto endosome trafficking. Nat. Cell Biol. 11, 1049–1051 (2009).

  39. 39.

    et al. The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing. EMBO J. 32, 1115–1127 (2013).

  40. 40.

    et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504 (2003).

  41. 41.

    , & BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics 21, 3448–3449 (2005).

Download references


We dedicate this work to the memory of MIT police officer Sean Collier who valiantly gave his life for the protection of the MIT community. We would like to thank P. Lobel and L. Huang from Rutgers University for NPC1 primary and immortalized cell lines. We wish to thank W. Salmon and N. Watson of the Whitehead Institute Core facility at MIT for help with confocal imaging. We would also like to thank D. Brown (Harvard MGH), K. Whitehead, R. Bogorad (MIT) and H. Yin (MIT) for healthy discussion. We would also like to thank J. Maraganore (Alnylam) and M. Invernale (MIT) for critical reading of the manuscript. Special thanks to D. Alakhova at University of Nebraska Medical Center for her help with graphic design. We would also like to thank Alnylam Pharmaceuticals, Control release grant EB000244 for funding. This work was supported by the National Heart, Lung, and Blood Institute, US National Institutes of Health, as a Program of Excellence in Nanotechnology (PEN) Award, Contract #HHSN268201000045C.

Author information


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

    • Gaurav Sahay
    • , Emmanouil Karagiannis
    • , Kevin Love
    • , Delai Chen
    • , Avi Schroeder
    • , Robert Langer
    •  & Daniel G Anderson
  2. Alnylam Pharmaceuticals, Cambridge, Massachusetts, USA.

    • William Querbes
    •  & Christopher Zurenko
  3. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Christopher Alabi
    • , Ahmed Eltoukhy
    • , Kevin Love
    • , Avi Schroeder
    • , Robert Langer
    •  & Daniel G Anderson
  4. Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Sovan Sarkar
    • , Roberto Zoncu
    •  & Yosef Buganim
  5. Harvard-MIT Division of Health Science and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Robert Langer
    •  & Daniel G Anderson
  6. Institute for Medical Engineering and Science, Cambridge, Massachusetts, USA.

    • Robert Langer
    •  & Daniel G Anderson


  1. Search for Gaurav Sahay in:

  2. Search for William Querbes in:

  3. Search for Christopher Alabi in:

  4. Search for Ahmed Eltoukhy in:

  5. Search for Sovan Sarkar in:

  6. Search for Christopher Zurenko in:

  7. Search for Emmanouil Karagiannis in:

  8. Search for Kevin Love in:

  9. Search for Delai Chen in:

  10. Search for Roberto Zoncu in:

  11. Search for Yosef Buganim in:

  12. Search for Avi Schroeder in:

  13. Search for Robert Langer in:

  14. Search for Daniel G Anderson in:


G.S., R.L. and D.G.A. conceived the idea, G.S., W.Q., R.L. and D.G.A. designed research, G.S., W.Q. and C.Z. performed high-throughput microscopy. G.S., C.A., A.E., K.L., D.C. and A.S. designed LNPs and their probes, G.S., C.A., A.E., S.S. and Y.B. performed studies with NPC1 deficient and competent cells, G.S. and S.S. performed studies with autophagy based studies, G.S. and R.Z. performed TIRF microscopy, G.S. and E.K. performed systems biology, G.S., W.Q., R.L. and D.G.A. wrote the manuscript.

Competing interests

R.L. is a shareholder and member of the Scientific Advisory Board of Alnylam. D.G.A. is a consultant with Alnylam. R.L and D.G.A have research grants sponsored by Alnylam. W.Q. and C.Z. are employed by Alnylam.

Corresponding author

Correspondence to Daniel G Anderson.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–11, Supplementary Table 1 and Supplementary Data


  1. 1.

    Supplementary Movie 1

    LNP trafficking at the cell membrane (LNP Alone.avi)

  2. 2.

    Supplementary Movie 2

    Effect of proton pump inhibitor on LNP trafficking at the cell membrane (LNP in presence of baf.avi)

About this article

Publication history






Further reading