Lipid Nanoparticle Formulation and Characterization

LNPs were prepared using NanoAssemblr Benchtop Instrument (Precision Nanosystems Inc., Vancouver, Canada) according to a previously described method (26 (link)). The lipid components (ionizable lipid, DOPE, cholesterol, and PEG lipid at 26.5:20:52:1.5 molar ratio) were dissolved in ethanol, and RNAs (Cas9 mRNA/sgRNA at 1:1 weight ratio) were dissolved in 10 mM citrate buffer (pH 3). The final ionizable lipid:RNA weight ratio was 10:1, and the final volume ratio was 1:3. Then, LNPs were formulated by microfluidic mixing of the prepared solutions at a 12 ml/min flow rate. The resulting LNPs were dialyzed against 1X phosphate-buffered saline (PBS) with 3500–molecular weight cutoff dialysis cassettes (Life Technologies) for 16 hours to exchange buffer. To characterize the prepared LNPs, dynamic light scattering was used to confirm the size, PDI, and zeta potential of LNPs. The encapsulation efficiency of RNAs was measured by Quant-iTTM Ribogreen Assay (Life Technologies).

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Han J.P., Kim M., Choi B.S., Lee J.H., Lee G.S., Jeong M., Lee Y., Kim E.A., Oh H.K., Go N., Lee H., Lee K.J., Kim U.G., Lee J.Y., Kim S., Chang J., Lee H., Song D.W, & Yeom S.C. (2022). In vivo delivery of CRISPR-Cas9 using lipid nanoparticles enables antithrombin gene editing for sustainable hemophilia A and B therapy. Science Advances, 8(3), eabj6901.

Publication 2022

NanoAssemblr Benchtop Instrument Quant-iTTM Ribogreen Assay

Assay Buffer Cholesterol Citrate Dialysis Ethanol Lipid Molar Mrna Phosphate Rnas Saline

Corresponding Organization : ToolGen (South Korea)

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Variable analysis

independent variables

Flow rate of microfluidic mixing (12 ml/min)

dependent variables

Size of LNPs
Polydispersity index (PDI) of LNPs
Zeta potential of LNPs
Encapsulation efficiency of RNAs

control variables

Lipid components (ionizable lipid, DOPE, cholesterol, and PEG lipid) at 26.5:20:52:1.5 molar ratio
Final ionizable lipid:RNA weight ratio (10:1)
Final volume ratio (1:3)
Dialysis against 1X phosphate-buffered saline (PBS) for 16 hours

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