Fusion-dependent formation of lipid nanoparticles containing macromolecular payloads
Open accessAbstract
The success of Onpattro™ (patisiran) clearly demonstrates the utility of lipid nanoparticle (LNP) systems for enabling gene therapies. These systems are composed of ionizable cationic lipids, phospholipid, cholesterol, and polyethylene glycol (PEG)-lipids, and are produced through rapid-mixing of an ethanolic-lipid solution with an acidic aqueous solution followed by dialysis into neutralizing buffer. A detailed understanding of the mechanism of LNP formation is crucial to improving LNP design. Here we use cryogenic transmission electron microscopy and fluorescence techniques to further demonstrate that LNP are formed through the fusion of precursor, pH-sensitive liposomes into large electron-dense core structures as the pH is neutralized. Next, we show that the fusion process is limited by the accumulation of PEG-lipid on the emerging particle. Finally, we show that the fusion-dependent mechanism of formation also applies to LNP containing macromolecular payloads including mRNA, DNA vectors, and gold nanoparticles.
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During LNP formulation, low pH protonates the ionizable lipid's amine, enabling electrostatic complexation of positively charged lipids and negatively charged mRNA; dialysis or ultrafiltration then neutralizes pH, resulting in uncharged solid-core LNPs that densely package mRNA.
“During the mixing step, the pH of the formulation is kept low to protonate the ionizable lipid's amine function, enabling electrostatic complexation of the now positively charged lipids and the negatively charged mRNA. A dialysis or ultrafiltration is then performed to neutralize the pH, resulting in uncharged, solid-core LNPs that densely package the mRNA [14]”
From The dawn of mRNA vaccines: the COVID-19 case (2021) · cites this paper as [14]
During the dialysis step, particles undergo fusion as the pH is neutralized, generating the final LNP system.
“Next, during the dialysis step these particles undergo fusion as the pH is neutralized, which generates the final LNP system.”
From Three decades of messenger RNA vaccine development (2019) · cites this paper as [112]
PEG-lipid content dictates the eventual LNP size by exerting repulsive forces at the particle surface that limit further fusion.
“Furthermore, they demonstrated that the PEG-lipid content dictates the eventual LNP size by exerting repulsive forces at the particle surface that limits further fusion.”
From Three decades of messenger RNA vaccine development (2019) · cites this paper as [112]
In the rapid-mixing step at acidic pH, empty small liposomal structures and loaded larger electron-dense particles are formed.
“They proposed that in the rapid-mixing step at acidic pH, (”
From Three decades of messenger RNA vaccine development (2019) · cites this paper as [112]
The mechanism of formation of ionizable LNP systems with different nucleic acid payloads has recently been elucidated by Kulkarni and colleagues.
“The mechanism of the formation of these ionizable LNP systems (with different nucleic acid payloads) has recently been elucidated by Kulkarni and colleagues [111,112].”
From Three decades of messenger RNA vaccine development (2019) · cites this paper as [112]
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“Intriguingly, mRNA-LNPs are known to form both spherical and highly nonspherical morphologies (9,11,16,18,19), but the origins and practical implications of these morphological variants”
BackgroundNanomedicine-Based Approaches for mRNA Delivery
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- Authors
- Kulkarni, J. A., Witzigmann, D., Leung, J., van der Meel, R., Zaifman, J., Darjuan, M. M., Grisch-Chan, H. M., Thöny, B., Tam, Y. Y. C., & Cullis, P. R.
- Affiliations
- University of British Columbia (CA)
- Department of Biochemistry and Molecular Biology
- Division of Metabolism
- University Children's Hospital Zurich (CH)
- Corresponding author
- Kulkarni, Jayesh A.
- Cullis, Pieter R.
- ORCID
- Venue
- Nanoscale
- Publisher
- Royal Society of Chemistry (RSC)
- Published
- 2019
- Volume
- 11
- Issue
- 18
- Pages
- 9023-9031
- ISSN
- 2040-3364 (ISSN-L)
- 2040-3372
- OpenAlex
- W2941514458
- Cited by
- 161
- References
- 25
- Citations by year
- 2026: 22 · 2025: 32 · 2024: 29 · 2023: 19 · 2022: 19 · 2021: 24 · 2020: 8 · 2019: 7
- FWCI
- 4.80
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Nanoscale (publishedVersion)
- PubMed (publishedVersion)
- TU/e Research Portal (publishedVersion, other-oa)
- Zurich Open Repository and Archive (University of Zurich) (acceptedVersion, other-oa)
- TU/e Research Portal (submittedVersion)
- Zurich Open Repository and Archive (University of Zurich) (submittedVersion, other-oa)
- TU/e Research Portal (submittedVersion, other-oa)
- Zurich Open Repository and Archive (University of Zurich)
- Funders
- Canadian Institutes of Health Research (FDN 148469)
- British Columbia Innovation Council (Ignite)
- Nederlandse Organisatie voor Wetenschappelijk Onderzoek (VENI 14385)
- Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Early Postdoc.Mobility Fellowship 174975, Sinergia 180257)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Physical Sciences › Materials Science › Biomaterials › Nanoparticle-Based Drug Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Lipid Membrane Structure and Behavior
- Keywords
- lipid nanoparticles, liposome fusion, pH-sensitive liposomes, PEG-lipids, ionizable cationic lipids, gene therapy, mRNA delivery, DNA vectors, gold nanoparticles, cryo-TEM
- MeSH terms
- Hydrogen-Ion Concentration, Lipids, Liposomes, Particle Size, Polyethylene Glycols, RNA, Messenger, Genetic Therapy, Cryoelectron Microscopy, RNA, Small Interfering, Macromolecular Substances, Nanoparticles
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