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Effects of Serum Incubation on Lipid Nanoparticle PEG Shedding, mRNA Retention, and Membrane Interactions

Niederkofler, Simon ; Parkkila, Petteri ; Aliakbarinodehi, Nima ; Sasanian, Nima ; Emilsson, Gustav ; Ulkoski, David ; Ferreira, Celso J.O. ; Galenkamp, Nicole Stéphanie LU orcid ; Silva, Bruno F.B. and Lundberg, Dan , et al. (2025) In ACS Applied Materials and Interfaces 17(47). p.64219-64231
Abstract

Lipid nanoparticles (LNPs) are widely used for RNA delivery, but their efficiency remains limited, largely due to poor endosomal escape. Upon administration, proteins bind to the surface of the LNPs, influencing cellular uptake and potentially altering their interfacial properties. Such alterations may also affect their interaction with endosomal membranes, thus influencing the critical endosomal escape step. Using fluorescence microscopy imaging with single-LNP resolution, this study investigates how incubation in 10% fetal bovine serum alters the PEG modification and mRNA content of LNPs, as well as how serum incubation-induced alterations influence the interaction between LNPs and an anionic supported lipid bilayer (SLB), serving as... (More)

Lipid nanoparticles (LNPs) are widely used for RNA delivery, but their efficiency remains limited, largely due to poor endosomal escape. Upon administration, proteins bind to the surface of the LNPs, influencing cellular uptake and potentially altering their interfacial properties. Such alterations may also affect their interaction with endosomal membranes, thus influencing the critical endosomal escape step. Using fluorescence microscopy imaging with single-LNP resolution, this study investigates how incubation in 10% fetal bovine serum alters the PEG modification and mRNA content of LNPs, as well as how serum incubation-induced alterations influence the interaction between LNPs and an anionic supported lipid bilayer (SLB), serving as a simplistic mimic for the anionic lipid membrane of late endosomes. We demonstrate that serum incubation leads to the desorption of PEG-modified lipids and a significant release of mRNA cargo from the LNPs. PEG shedding occurred consistently with a half-life time of around 10 min, while mRNA release displayed higher variability between individual LNPs. We also observed that serum preincubation enhanced attractive interactions between tethered LNPs and the anionic SLB at physiological pH 7.4, and fusion of LNPs with the anionic SLB upon pH reduction was more efficient for serum-preincubated LNPs than for their pristine counterparts, particularly during moderate acidification from pH 6.5 to 6.0. This enhanced fusion efficiency may be attributed to a reduced steric hindrance from PEG-lipids following serum preincubation. The findings highlight that serum-induced modifications enhance LNP fusion efficiency with an endosomal membrane mimic while potentially compromising mRNA retention, thus balancing the overall efficacy of LNP-assisted mRNA delivery.

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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
endosomal escape, endosomal membrane mimic, lipid nanoparticle (LNP), mRNA delivery, PEG shedding, protein corona
in
ACS Applied Materials and Interfaces
volume
17
issue
47
pages
13 pages
publisher
The American Chemical Society (ACS)
external identifiers
  • scopus:105022914494
  • pmid:41234148
ISSN
1944-8244
DOI
10.1021/acsami.5c17052
language
English
LU publication?
yes
additional info
Publisher Copyright: © 2025 The Authors. Published by American Chemical Society
id
47608967-0bfc-48b4-8d5d-499a89ce1a98
date added to LUP
2026-01-19 11:35:38
date last changed
2026-01-19 11:36:43
@article{47608967-0bfc-48b4-8d5d-499a89ce1a98,
  abstract     = {{<p>Lipid nanoparticles (LNPs) are widely used for RNA delivery, but their efficiency remains limited, largely due to poor endosomal escape. Upon administration, proteins bind to the surface of the LNPs, influencing cellular uptake and potentially altering their interfacial properties. Such alterations may also affect their interaction with endosomal membranes, thus influencing the critical endosomal escape step. Using fluorescence microscopy imaging with single-LNP resolution, this study investigates how incubation in 10% fetal bovine serum alters the PEG modification and mRNA content of LNPs, as well as how serum incubation-induced alterations influence the interaction between LNPs and an anionic supported lipid bilayer (SLB), serving as a simplistic mimic for the anionic lipid membrane of late endosomes. We demonstrate that serum incubation leads to the desorption of PEG-modified lipids and a significant release of mRNA cargo from the LNPs. PEG shedding occurred consistently with a half-life time of around 10 min, while mRNA release displayed higher variability between individual LNPs. We also observed that serum preincubation enhanced attractive interactions between tethered LNPs and the anionic SLB at physiological pH 7.4, and fusion of LNPs with the anionic SLB upon pH reduction was more efficient for serum-preincubated LNPs than for their pristine counterparts, particularly during moderate acidification from pH 6.5 to 6.0. This enhanced fusion efficiency may be attributed to a reduced steric hindrance from PEG-lipids following serum preincubation. The findings highlight that serum-induced modifications enhance LNP fusion efficiency with an endosomal membrane mimic while potentially compromising mRNA retention, thus balancing the overall efficacy of LNP-assisted mRNA delivery.</p>}},
  author       = {{Niederkofler, Simon and Parkkila, Petteri and Aliakbarinodehi, Nima and Sasanian, Nima and Emilsson, Gustav and Ulkoski, David and Ferreira, Celso J.O. and Galenkamp, Nicole Stéphanie and Silva, Bruno F.B. and Lundberg, Dan and Jing, Yujia and Lindfors, Lennart and Agnarsson, Björn and Höök, Fredrik}},
  issn         = {{1944-8244}},
  keywords     = {{endosomal escape; endosomal membrane mimic; lipid nanoparticle (LNP); mRNA delivery; PEG shedding; protein corona}},
  language     = {{eng}},
  month        = {{11}},
  number       = {{47}},
  pages        = {{64219--64231}},
  publisher    = {{The American Chemical Society (ACS)}},
  series       = {{ACS Applied Materials and Interfaces}},
  title        = {{Effects of Serum Incubation on Lipid Nanoparticle PEG Shedding, mRNA Retention, and Membrane Interactions}},
  url          = {{http://dx.doi.org/10.1021/acsami.5c17052}},
  doi          = {{10.1021/acsami.5c17052}},
  volume       = {{17}},
  year         = {{2025}},
}