On the formation and morphology of lipid nanoparticles containing Ionizable cationic lipids and siRNA

Jayesh A. Kulkarni, Maria M. Darjuan, Joanne E. Mercer, Sam Chen, Roy van der Meel, Jenifer L. Thewalt, Yuen Yi C Tam, Pieter R. Cullis

Onderzoeksoutput: Bijdrage aan tijdschriftTijdschriftartikelAcademicpeer review

10 Citaties (Scopus)

Uittreksel

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 Ka 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.

TaalEngels
Pagina's4787-4795
Aantal pagina's9
TijdschriftACS Nano
Volume12
Nummer van het tijdschrift5
DOI's
StatusGepubliceerd - 22 mei 2018
Extern gepubliceerdJa

Vingerafdruk

Lipids
Small Interfering RNA
lipids
Nanoparticles
nanoparticles
Monolayers
Buffers
Ethanol
ethyl alcohol
buffers
Cholesterol
cholesterol
RNA
genes
Cryogenics
Polyethylene glycols
cryogenics
Oils

Citeer dit

Kulkarni, J. A., Darjuan, M. M., Mercer, J. E., Chen, S., van der Meel, R., Thewalt, J. L., ... Cullis, P. R. (2018). On the formation and morphology of lipid nanoparticles containing Ionizable cationic lipids and siRNA. ACS Nano, 12(5), 4787-4795. DOI: 10.1021/acsnano.8b01516
Kulkarni, Jayesh A. ; Darjuan, Maria M. ; Mercer, Joanne E. ; Chen, Sam ; van der Meel, Roy ; Thewalt, Jenifer L. ; Tam, Yuen Yi C ; Cullis, Pieter R./ On the formation and morphology of lipid nanoparticles containing Ionizable cationic lipids and siRNA. In: ACS Nano. 2018 ; Vol. 12, Nr. 5. blz. 4787-4795
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abstract = "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 Ka 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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Kulkarni, JA, Darjuan, MM, Mercer, JE, Chen, S, van der Meel, R, Thewalt, JL, Tam, YYC & Cullis, PR 2018, 'On the formation and morphology of lipid nanoparticles containing Ionizable cationic lipids and siRNA' ACS Nano, vol. 12, nr. 5, blz. 4787-4795. DOI: 10.1021/acsnano.8b01516

On the formation and morphology of lipid nanoparticles containing Ionizable cationic lipids and siRNA. / Kulkarni, Jayesh A.; Darjuan, Maria M.; Mercer, Joanne E.; Chen, Sam; van der Meel, Roy; Thewalt, Jenifer L.; Tam, Yuen Yi C; Cullis, Pieter R.

In: ACS Nano, Vol. 12, Nr. 5, 22.05.2018, blz. 4787-4795.

Onderzoeksoutput: Bijdrage aan tijdschriftTijdschriftartikelAcademicpeer review

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AU - Kulkarni,Jayesh A.

AU - Darjuan,Maria M.

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AU - Chen,Sam

AU - van der Meel,Roy

AU - Thewalt,Jenifer L.

AU - Tam,Yuen Yi C

AU - Cullis,Pieter R.

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N2 - 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 Ka 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.

AB - 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 Ka 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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Kulkarni JA, Darjuan MM, Mercer JE, Chen S, van der Meel R, Thewalt JL et al. On the formation and morphology of lipid nanoparticles containing Ionizable cationic lipids and siRNA. ACS Nano. 2018 mei 22;12(5):4787-4795. Beschikbaar vanaf, DOI: 10.1021/acsnano.8b01516