On the formation and morphology of lipid nanoparticles containing ionizable cationic lipids and siRNA
Open accessAbstract
Lipid nanoparticles (LNPs) containing short interfering RNA (LNP-siRNA) and optimized ionizable cationic lipids are now clinically validated systems for silencing disease-causing genes in hepatocytes following intravenous administration. However, the mechanism of formation and certain structural features of LNP-siRNA remain obscure. These systems are formed from lipid mixtures (cationic lipid, distearoylphosphatidylcholine, cholesterol, and PEG-lipid) dissolved in ethanol that is rapidly mixed with siRNA in aqueous buffer at a pH (pH 4) where the ionizable lipid is positively charged. The resulting dispersion is then dialyzed against a normal saline buffer to remove residual ethanol and raise the pH to 7.4 (above the p K a of the cationic lipid) to produce the finished LNP-siRNA systems. Here we provide cryogenic transmission electron microscopy (cryo-TEM) and X-ray evidence that the complexes formed between siRNA and ionizable lipid at pH 4 correspond to tightly packed bilayer structures with siRNA sandwiched between closely apposed monolayers. Further, it is shown that ionizable lipid not complexed to siRNA promotes formation of very small vesicular structures at pH 4 that coalesce to form larger LNP structures with amorphous electron dense cores at pH 7.4. A mechanism of formation of LNP-siRNA systems is proposed whereby siRNA is first sandwiched between closely apposed lipid monolayers at pH 4 and subsequently trapped in these structures as the pH is raised to 7.4, whereas ionizable lipid not interacting with siRNA moves from bilayer structure to adopt an amorphous oil phase located in the center of the LNP as the pH is raised. This model is discussed in terms of previous hypotheses and potential relevance to the design of LNP-siRNA systems.
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A key aspect of LNPs is the presence of lipids in the core, although water is also present to some extent.
“A key aspect of LNPs and the characteristic that makes them different from liposomes (spherical vesicles with at least one lipid bilayer and an aqueous core) is the presence of lipids in the core, although data from several studies indicate that water is also present to some extent (Yanez Arteta et al., 2018, Kulkarni et al., 2018, Brader et al., 2021).”
From mRNA-lipid nanoparticle COVID-19 vaccines: structure and stability (2021) · cites this paper as Kulkarni et al., 2018
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 [111]
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 [111]
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 [111]
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 [111]
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- Authors
- Kulkarni, J. A., Darjuan, M. M., Mercer, J. E., Chen, S., van der Meel, R., Thewalt, J. L., Tam, Y. Y. C., & Cullis, P. R.
- Affiliations
- University of British Columbia (CA)
- Simon Fraser University (CA)
- Department of Biochemistry and Molecular Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada
- Corresponding author
- Cullis, Pieter R.
- Venue
- ACS Nano
- Publisher
- American Chemical Society (ACS)
- Published
- 2018-04-03
- Volume
- 12
- Issue
- 5
- Pages
- 4787-4795
- ISSN
- 1936-0851 (ISSN-L)
- 1936-086X
- OpenAlex
- W2795918326
- Cited by
- 663
- References
- 24
- Citations by year
- 2026: 112 · 2025: 146 · 2024: 109 · 2023: 89 · 2022: 90 · 2021: 59 · 2020: 31 · 2019: 20 · 2018: 5
- FWCI
- 10.22
- Type
- journal article
- Language
- en
- Open access
- green
- dspace.library.uu.nl
- Licence
- other-oa
- Locations
- ACS Nano (publishedVersion)
- PubMed (publishedVersion)
- TU/e Research Portal (submittedVersion)
- Utrecht University Repository (Utrecht University) (submittedVersion, other-oa)
- Utrecht University Repository (Utrecht University) (submittedVersion)
- Funders
- Canadian Institutes of Health Research (FDN 148469)
- British Columbia Innovation Council
- Natural Sciences and Engineering Research Council of Canada (STPGP/463247-2014)
- Nederlandse Organisatie voor Wetenschappelijk Onderzoek (14385)
- 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, siRNA, ionizable cationic lipids, lipid bilayer structure, distearoylphosphatidylcholine, PEG-lipids, cholesterol, amorphous core, vesicle coalescence, cryo-TEM, X-ray diffraction, gene silencing, intravenous delivery
- MeSH terms
- Cholesterol, Hydrogen-Ion Concentration, Lipids, Particle Size, Phosphatidylcholines, Polyethylene Glycols, Surface Properties, Gene Transfer Techniques, RNA, Small Interfering, Phase Transition, Nanoparticles
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