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Cellular and biophysical barriers to lipid nanoparticle mediated delivery of RNA to the cytosol

Johansson, Johanna M. LU ; Du Rietz, Hampus LU orcid ; Hedlund, Hampus LU ; Eriksson, Hanna C. LU orcid ; Oude Blenke, Erik ; Pote, Aditya ; Harun, Said ; Nordenfelt, Pontus LU orcid ; Lindfors, Lennart and Wittrup, Anders LU (2025) In Nature Communications 16(1).
Abstract
Lipid nanoparticles (LNPs) are clinically approved for mRNA-based vaccines and liver-targeted siRNA delivery. However, poor nucleic acid delivery efficiency limits their application in extrahepatic tissues and tumors. Here, using live-cell and super-resolution microscopy, we identify multiple distinct steps of inefficiencies in the cytosolic delivery of both siRNA and mRNA cargoes. Membrane damages marked by galectin recruitment are conducive to cytosolic RNA release, whereas membrane perturbations recruiting the ESCRT machinery do not permit endosomal escape. Notably, only a small fraction of RNA is released from galectin-marked endosomes and, unexpectedly, many damaged endosomes contain no detectable RNA cargo. Using LNPs with both... (More)
Lipid nanoparticles (LNPs) are clinically approved for mRNA-based vaccines and liver-targeted siRNA delivery. However, poor nucleic acid delivery efficiency limits their application in extrahepatic tissues and tumors. Here, using live-cell and super-resolution microscopy, we identify multiple distinct steps of inefficiencies in the cytosolic delivery of both siRNA and mRNA cargoes. Membrane damages marked by galectin recruitment are conducive to cytosolic RNA release, whereas membrane perturbations recruiting the ESCRT machinery do not permit endosomal escape. Notably, only a small fraction of RNA is released from galectin-marked endosomes and, unexpectedly, many damaged endosomes contain no detectable RNA cargo. Using LNPs with both fluorescently labeled ionizable lipid and RNA, we show that these components segregate during endosomal sorting – both within single endosomes and across endosomal compartments. Finally, we visualize localized ionizable lipid enrichment in endosomal membranes and membrane damage in direct proximity to siRNA-LNPs tethered to luminal vesicle membranes. Taken together, our findings reveal multiple mechanistic barriers limiting intracellular RNA delivery by LNPs. (Less)
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publishing date
type
Contribution to journal
publication status
published
subject
in
Nature Communications
volume
16
issue
1
article number
5354
publisher
Nature Publishing Group
ISSN
2041-1723
DOI
10.1038/s41467-025-60959-z
language
English
LU publication?
yes
id
159d1b1a-e9f3-4518-b217-8ed7f938c414
date added to LUP
2025-07-01 13:45:12
date last changed
2025-07-01 13:48:12
@article{159d1b1a-e9f3-4518-b217-8ed7f938c414,
  abstract     = {{Lipid nanoparticles (LNPs) are clinically approved for mRNA-based vaccines and liver-targeted siRNA delivery. However, poor nucleic acid delivery efficiency limits their application in extrahepatic tissues and tumors. Here, using live-cell and super-resolution microscopy, we identify multiple distinct steps of inefficiencies in the cytosolic delivery of both siRNA and mRNA cargoes. Membrane damages marked by galectin recruitment are conducive to cytosolic RNA release, whereas membrane perturbations recruiting the ESCRT machinery do not permit endosomal escape. Notably, only a small fraction of RNA is released from galectin-marked endosomes and, unexpectedly, many damaged endosomes contain no detectable RNA cargo. Using LNPs with both fluorescently labeled ionizable lipid and RNA, we show that these components segregate during endosomal sorting – both within single endosomes and across endosomal compartments. Finally, we visualize localized ionizable lipid enrichment in endosomal membranes and membrane damage in direct proximity to siRNA-LNPs tethered to luminal vesicle membranes. Taken together, our findings reveal multiple mechanistic barriers limiting intracellular RNA delivery by LNPs.}},
  author       = {{Johansson, Johanna M. and Du Rietz, Hampus and Hedlund, Hampus and Eriksson, Hanna C. and Oude Blenke, Erik and Pote, Aditya and Harun, Said and Nordenfelt, Pontus and Lindfors, Lennart and Wittrup, Anders}},
  issn         = {{2041-1723}},
  language     = {{eng}},
  number       = {{1}},
  publisher    = {{Nature Publishing Group}},
  series       = {{Nature Communications}},
  title        = {{Cellular and biophysical barriers to lipid nanoparticle mediated delivery of RNA to the cytosol}},
  url          = {{http://dx.doi.org/10.1038/s41467-025-60959-z}},
  doi          = {{10.1038/s41467-025-60959-z}},
  volume       = {{16}},
  year         = {{2025}},
}