Fusion-dependent formation of lipid nanoparticles containing macromolecular payloads

Jayesh A. Kulkarni; Dominik Witzigmann; Jerry Leung; Roy van der Meel; Josh Zaifman; Maria M. Darjuan; Hiu Man Grisch-Chan; Beat Thöny; Yuen Yi C. Tam; Pieter R. Cullis

doi:10.1039/C9NR02004G

Fusion-dependent formation of lipid nanoparticles containing macromolecular payloads

Jayesh A. Kulkarni (Corresponding author), Dominik Witzigmann, Jerry Leung, Roy van der Meel, Josh Zaifman, Maria M. Darjuan, Hiu Man Grisch-Chan, Beat Thöny, Yuen Yi C. Tam, Pieter R. Cullis (Corresponding author)

Precision Medicine

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Abstract

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.

Original language	English
Pages (from-to)	9023-9031
Number of pages	9
Journal	Nanoscale
Volume	11
Issue number	18
Early online date	22 Apr 2019
DOIs	https://doi.org/10.1039/C9NR02004G
Publication status	Published - 14 May 2019

Funding

This work was supported by Foundation grant (FDN 148469) from the Canadian Institutes of Health Research, and a British Columbia Innovation Council Ignite grant. DW acknowledges the support by the Swiss National Science Foundation (SNF, Early Postdoc.Mobility Fellowship, Grant No. 174975). RvdM is supported by a VENI Fellowship (# 14385) from the Netherlands Organization for Scientific Research (NWO). HMGC is supported by the SNF Sinergia grant #180257 (to BT). The authors acknowledge N. Rimann for support with mcDNA production.

Keywords

Cryoelectron Microscopy
Genetic Therapy/methods
Hydrogen-Ion Concentration
Lipids/chemistry
Liposomes/chemistry
Macromolecular Substances/chemistry
Nanoparticles/chemistry
Particle Size
Polyethylene Glycols/chemistry
RNA, Messenger/chemistry
RNA, Small Interfering/chemistry

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10.1039/C9NR02004G

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title = "Fusion-dependent formation of lipid nanoparticles containing macromolecular payloads",

abstract = "The success of Onpattro{\texttrademark} (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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AU - Darjuan, Maria M.

AU - Grisch-Chan, Hiu Man

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AU - Tam, Yuen Yi C.

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AB - 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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Fusion-dependent formation of lipid nanoparticles containing macromolecular payloads

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