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Image-based analysis of lipid nanoparticle–mediated siRNA delivery, intracellular trafficking and endosomal escape

Nature Biotechnology volume 31, pages 638–646 (2013)Cite this article

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Abstract

Delivery of short interfering RNAs (siRNAs) remains a key challenge in the development of RNA interference (RNAi) therapeutics. A better understanding of the mechanisms of siRNA cellular uptake, intracellular transport and endosomal release could critically contribute to the improvement of delivery methods. Here we monitored the uptake of lipid nanoparticles (LNPs) loaded with traceable siRNAs in different cell types in vitro and in mouse liver by quantitative fluorescence imaging and electron microscopy. We found that LNPs enter cells by both constitutive and inducible pathways in a cell type-specific manner using clathrin-mediated endocytosis as well as macropinocytosis. By directly detecting colloidal-gold particles conjugated to siRNAs, we estimated that escape of siRNAs from endosomes into the cytosol occurs at low efficiency (1–2%) and only during a limited window of time when the LNPs reside in a specific compartment sharing early and late endosomal characteristics. Our results provide insights into LNP-mediated siRNA delivery that can guide development of the next generation of delivery systems for RNAi therapeutics.

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Figure 1: LNP-siRNA, LNP-siRNA-alexa647 and LNP-siRNA-gold have similar size and knock-down efficiency.
Figure 2: LNP uptake is mediated by multiple Dynamin-dependent pathways.
Figure 3: LNP uptake in HeLa cells induces the formation of a hybrid early-late endosomal compartment through recruitment of Rabankyrin-5.
Figure 4: Ultrastructural analysis of LNP in vitro trafficking.
Figure 5: In vivo analysis of LNP bioavailability and intracellular trafficking.
Figure 6: In vitro and in vivo cytosolic release of siRNAs occurs in a specific stage of LNP intracellular trafficking, most probably within the early endosome.

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References

  1. Vaishnaw, A.K. et al. A status report on RNAi therapeutics. Silence 1, 14 (2010).

    Article  Google Scholar 

  2. Czech, B. & Hannon, G.J. Small RNA sorting: matchmaking for Argonautes. Nat. Rev. Genet. 12, 19–31 (2011).

    Article  CAS  Google Scholar 

  3. Sliva, K. & Schnierle, B.S. Selective gene silencing by viral delivery of short hairpin RNA. Virol. J. 7, 248 (2010).

    Article  Google Scholar 

  4. Shim, M.S. & Kwon, Y.J. Efficient and targeted delivery of siRNA in vivo. FEBS J. 277, 4814–4827 (2010).

    Article  CAS  Google Scholar 

  5. Zhang, S., Zhao, Y., Zhi, D. & Zhang, S. Non-viral vectors for the mediation of RNAi. Bioorg. Chem. 40, 10–18 (2012).

    Article  Google Scholar 

  6. Semple, S.C. et al. Rational design of cationic lipids for siRNA delivery. Nat. Biotechnol. 28, 172–176 (2010).

    Article  CAS  Google Scholar 

  7. Jayaraman, M. et al. Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angew. Chem. Int. Edn Engl. 51, 8529–8533 (2012).

    Article  CAS  Google Scholar 

  8. Frank-Kamenetsky, M. et al. Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates. Proc. Natl. Acad. Sci. USA 105, 11915–11920 (2008).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Akinc, A. et al. A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat. Biotechnol. 26, 561–569 (2008).

    Article  CAS  Google Scholar 

  11. Zeigerer, A. et al. Rab5 is necessary for the biogenesis of the endolysosomal system in vivo. Nature 485, 465–470 (2012).

    Article  CAS  Google Scholar 

  12. Trial to Evaluate Safety and Tolerability of ALN-PCS02 in Subjects With Elevated LDL-Cholesterol (LDL-C). NCT01437059 (Registry of Federally and Privately Supported Clinical Trials, U.S. National Institutes of Health, 2011.

  13. Trial to Evaluate Safety, Tolerability, and Parmacokinetics of ALN-TTR02 in Healthy Volunteer Subjects. NCT01559077 (Registry of Federally and Privately Supported Clinical Trials, US National Institutes of Health, 2012.

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

    Article  CAS  Google Scholar 

  15. Basha, G. 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).

    Article  CAS  Google Scholar 

  16. Tamura, A., Oishi, M. & Nagasaki, Y. Enhanced cytoplasmic delivery of siRNA using a stabilized polyion complex based on PEGylated nanogels with a cross-linked polyamine structure. Biomacromolecules 10, 1818–1827 (2009).

    Article  CAS  Google Scholar 

  17. Akita, H. et al. Nanoparticles for ex vivo siRNA delivery to dendritic cells for cancer vaccines: programmed endosomal escape and dissociation. J. Control. Release 143, 311–317 (2010).

    Article  CAS  Google Scholar 

  18. Sakurai, Y. et al. Endosomal escape and the knockdown efficiency of liposomal-siRNA by the fusogenic peptide shGALA. Biomaterials 32, 5733–5742 (2011).

    Article  CAS  Google Scholar 

  19. Bramsen, J.B. et al. A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity. Nucleic Acids Res. 37, 2867–2881 (2009).

    Article  CAS  Google Scholar 

  20. Macia, E. et al. Dynasore, a cell-permeable inhibitor of dynamin. Dev. Cell 10, 839–850 (2006).

    Article  CAS  Google Scholar 

  21. Doherty, G.J. & McMahon, H.T. Mechanisms of endocytosis. Annu. Rev. Biochem. 78, 857–902 (2009).

    Article  CAS  Google Scholar 

  22. Schnatwinkel, C. et al. The Rab5 effector Rabankyrin-5 regulates and coordinates different endocytic mechanisms. PLoS Biol. 2, e261 (2004).

    Article  Google Scholar 

  23. Meier, O. et al. Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake. J. Cell Biol. 158, 1119–1131 (2002).

    Article  CAS  Google Scholar 

  24. Schmees, C. et al. Macropinocytosis of the PDGF beta-receptor promotes fibroblast transformation by H-RasG12V. Mol. Biol. Cell 23, 2571–2582 (2012).

    Article  CAS  Google Scholar 

  25. Gruenberg, J. & Maxfield, F.R. Membrane transport in the endocytic pathway. Curr. Opin. Cell Biol. 7, 552–563 (1995).

    Article  CAS  Google Scholar 

  26. Dominska, M. & Dykxhoorn, D.M. Breaking down the barriers: siRNA delivery and endosome escape. J. Cell Sci. 123, 1183–1189 (2010).

    Article  CAS  Google Scholar 

  27. Pei, Y. et al. Quantitative evaluation of siRNA delivery in vivo. RNA 16, 2553–2563 (2010).

    Article  CAS  Google Scholar 

  28. Maier, M. in 8th Annual Meeting of the Oligonucleotide Therapeutics Society, Boston. (2012).

  29. Wei, J. et al. RNA-induced silencing complex-bound small interfering RNA is a determinant of RNA interference-mediated gene silencing in mice. Mol. Pharmacol. 79, 953–963 (2011).

    Article  CAS  Google Scholar 

  30. Romberg, B., Hennink, W.E. & Storm, G. Sheddable coatings for long-circulating nanoparticles. Pharm. Res. 25, 55–71 (2008).

    Article  CAS  Google Scholar 

  31. Wheeler, J.J. et al. Stabilized plasmid-lipid particles: construction and characterization. Gene Ther. 6, 271–281 (1999).

    Article  CAS  Google Scholar 

  32. Tomas, M.I., Kucic, N., Mahmutefendic, H., Blagojevic, G. & Lucin, P. Murine cytomegalovirus perturbs endosomal trafficking of major histocompatibility complex class I molecules in the early phase of infection. J. Virol. 84, 11101–11112 (2010).

    Article  CAS  Google Scholar 

  33. Rink, J., Ghigo, E., Kalaidzidis, Y. & Zerial, M. Rab conversion as a mechanism of progression from early to late endosomes. Cell 122, 735–749 (2005).

    Article  CAS  Google Scholar 

  34. Poteryaev, D., Datta, S., Ackema, K., Zerial, M. & Spang, A. Identification of the switch in early-to-late endosome transition. Cell 141, 497–508 (2010).

    Article  CAS  Google Scholar 

  35. Landesman, Y. et al. In vivo quantification of formulated and chemically modified small interfering RNA by heating-in-Triton quantitative reverse transcription polymerase chain reaction (HIT qRT-PCR). Silence 1, 16 (2010).

    Article  Google Scholar 

  36. Lee, S.H., Choi, S.H., Kim, S.H. & Park, T.G. Thermally sensitive cationic polymer nanocapsules for specific cytosolic delivery and efficient gene silencing of siRNA: swelling induced physical disruption of endosome by cold shock. J. Control. Release 125, 25–32 (2008).

    Article  CAS  Google Scholar 

  37. Hafez, I.M., Maurer, N. & Cullis, P.R. On the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids. Gene Ther. 8, 1188–1196 (2001).

    Article  CAS  Google Scholar 

  38. Lewis, J. et al. In vivo silencing of alpha-synuclein using naked siRNA. Mol. Neurodegener. 3, 19 (2008).

    Article  Google Scholar 

  39. Hauser, P.V. et al. Novel siRNA delivery system to target podocytes in vivo. PLoS ONE 5, e9463 (2010).

    Article  Google Scholar 

  40. Xia, C.F., Boado, R.J. & Pardridge, W.M. Antibody-mediated targeting of siRNA via the human insulin receptor using avidin-biotin technology. Mol. Pharm. 6, 747–751 (2009).

    Article  CAS  Google Scholar 

  41. Valiunas, V. et al. Connexin-specific cell-to-cell transfer of short interfering RNA by gap junctions. J. Physiol. (Lond.) 568, 459–468 (2005).

    Article  CAS  Google Scholar 

  42. Wolfrum, C. et al. Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Nat. Biotechnol. 25, 1149–1157 (2007).

    Article  CAS  Google Scholar 

  43. Ohya, T. et al. Reconstitution of Rab- and SNARE-dependent membrane fusion by synthetic endosomes. Nature 459, 1091–1097 (2009).

    Article  CAS  Google Scholar 

  44. Starai, V.J., Jun, Y. & Wickner, W. Excess vacuolar SNAREs drive lysis and Rab bypass fusion. Proc. Natl. Acad. Sci. USA 104, 13551–13558 (2007).

    Article  CAS  Google Scholar 

  45. Andaloussi, S.E. et al. Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo. Nucleic Acids Res. 39, 3972–3987 (2011).

    Article  Google Scholar 

  46. Bartz, R. et al. Effective siRNA delivery and target mRNA degradation using an amphipathic peptide to facilitate pH-dependent endosomal escape. Biochem. J. 435, 475–487 (2011).

    Article  CAS  Google Scholar 

  47. Kobayashi, T. et al. The tetraspanin CD63/lamp3 cycles between endocytic and secretory compartments in human endothelial cells. Mol. Biol. Cell 11, 1829–1843 (2000).

    Article  CAS  Google Scholar 

  48. Damha, M.J. & Ogilvie, K.K. Oligoribonucleotide synthesis. The silyl-phosphoramidite method. Methods Mol. Biol. 20, 81–114 (1993).

    CAS  PubMed  Google Scholar 

  49. Soutschek, J. et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432, 173–178 (2004).

    Article  CAS  Google Scholar 

  50. Addepalli, H. et al. Modulation of thermal stability can enhance the potency of siRNA. Nucleic Acids Res. 38, 7320–7331 (2010).

    Article  CAS  Google Scholar 

  51. Manoharan, M., Kesavan, V. & Rajeev, K.G. Modified iRNA agents. US Patent 7,745,608 (2010).

Download references

Acknowledgements

We acknowledge T. Galvez and E. Perini for discussions and comments on the manuscript. We thank J.-M. Verbavatz and J. Peychl, respectively, for the management of the Electron Microscopy Facility and the Light Microscopy Facility. We thank A. Giner for cell culture, W. John and A. Muench-Wuttke from the Biomedical Service Facility for mouse care and injections, A. Pal and R. Villasenor for Rab5-GFP primary human fibroblasts and H-RasG12V cells preparation. We thank D. Butler, R. Kallanthottathil and M. Manoharan for advice on chemistry. This work was financially supported by the Max Planck Society (M.P.G.), the Virtual Liver initiative (http://www.virtual-liver.de) funded by the German Federal Ministry of Research and Education (BMBF), the DFG and Alnylam Pharmaceuticals. J.G. was supported by a grant from EMBO long-term fellowship.

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Authors and Affiliations

  1. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

    Jerome Gilleron, Anja Zeigerer, Giovanni Marsico, Undine Schubert, Kevin Manygoats, Sarah Seifert, Cordula Andree, Martin Stöter, Eugenio Fava, Marc Bickle, Yannis Kalaidzidis & Marino Zerial

  2. Alnylam Pharmaceuticals, Cambridge, Massachusetts, USA

    William Querbes, Anna Borodovsky, Hila Epstein-Barash, Ligang Zhang, Victor Koteliansky, Kevin Fitzgerald, Akin Akinc & Martin Maier

  3. German Center for Neurodegenerative Diseases, Bonn, Germany

    Eugenio Fava

  4. Moscow State University, Faculty of Bioengineering and Bioinformatics, Moscow, Russia

    Yannis Kalaidzidis

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Contributions

M.Z. conceived and directed the project. M.Z., J.G., V.K., A.Z., W.Q., A.A., A.B., K.F., M.M. and Y.K. designed the experiments. M.M. synthesized the siRNA-gold conjugates. L.Z. synthesized the siRNA-alexa647. H.E.-B. formulated LNPs. C.A. and U.S., under the supervision of J.G., A.Z., E.F. and M.B., performed the in vitro fluorescence microscopy staining. M.S. performed the OPERA automated image acquisitions. G.M., under the supervision of Y.K., provided the quantitative multi-parametric image analysis and the statistics. A.Z. and S.S. directed the mouse tail-vein injections and the liver perfusion. J.G. and A.Z. co-developed the staining procedures for the tissues sections. J.G. performed the sections, staining and imaging. J.G. helped by K.M. developed the quantitative electron microscopy. Y.K. developed the mathematical model of siRNA cytosolic release. G.M. developed the software for automated counting of gold number on electron microscopy images. M.Z., J.G., W.Q., Y.K. and M.M. wrote the manuscript.

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Correspondence to Marino Zerial.

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Competing interests

W.Q., A.B., H.H.-B., L.Z., V.K., K.F., A.A. and M.M. are Alnylam Pharmaceuticals employees; M.Z. received funding from and was a consultant with Alnylam Pharmaceuticals.

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Gilleron, J., Querbes, W., Zeigerer, A. et al. Image-based analysis of lipid nanoparticle–mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat Biotechnol 31, 638–646 (2013). https://doi.org/10.1038/nbt.2612

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