On the Formation and Morphology of Lipid Nanoparticles Containing Ionizable Cationic Lipids and siRNA

Apr 4, 2018ACS nano

How Lipid Nanoparticles with Ionizable Positive Lipids and siRNA Form and Shape Themselves

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Abstract

Cryogenic transmission electron microscopy and X-ray evidence reveal that complexes formed between siRNA and ionizable lipid at pH 4 correspond to tightly packed bilayer structures.

Lipid nanoparticles containing siRNA and optimized lipids are validated for silencing genes in liver cells after intravenous delivery.
At pH 4, siRNA is sandwiched between closely apposed lipid layers, forming a bilayer structure.
Ionizable lipid not associated with siRNA forms small vesicular structures at pH 4, which merge into larger particles at pH 7.4.
As pH increases to 7.4, the structure transforms, with siRNA trapped in the lipid layers and excess lipid adopting an amorphous core.
A proposed mechanism suggests initial formation at low pH leads to stabilization of siRNA within lipid structures upon pH elevation.

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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 Kof 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. a

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On the Formation and Morphology of Lipid Nanoparticles Containing Ionizable Cationic Lipids and siRNA

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