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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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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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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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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DOI: https://doi.org/10.1038/nbt.2612
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