Figure 1.

Ethanol Loading Formulation Process for LNP Containing Oligonucleotides Such as siRNA

Lipids dissolved in ethanol are rapidly mixed with oligonucleotides in aqueous buffer (pH 4). The head groups of the ionizable cationic lipids are indicated as blue, of the PEG-lipids as white lines and of DSPC as white circles. Cholesterol is indicated as a diamond orange symbol and acyl chains as yellow lines. On mixing, electrostatic interactions drive formation of an inverted micelle containing oligonucleotide (red zig-zag symbol) surrounded by predominantly cationic lipid that falls out of solution as the polarity is raised. If the mixing process occurs rapidly enough, these inverted micelles do not have time to aggregate but rather are coated with PEG-lipid, which precipitates at higher polarity to surround the inverted micelles.