*2.4. Preparation of RNA-Loaded LNPs*

An ethanol solution containing a pH-sensitive cationic lipid, a phospholipid, chol, and PEG-lipid at the indicated molar ratios was prepared at a total lipid concentration of 16 mM. The RNA was dissolved in 25 mM acetate buffer (pH 4.0) containing NaCl (0 to 400 mM). For MC3-LNPs, a fixed lipid composition of MC3:DSPC:chol:PEG-DMG = 50:10:40:1.5 (molar ratio) and acetate buffer without NaCl were used. LNPs were prepared by mixing the lipids in ethanol and an aqueous solution of mRNA using an iLiNP device at a total

flow rate (TFR) of 0.5 mL/min and RNA-to-lipid flow rate ratio (FRR) of 3. RNA-to-lipid ratio was adjusted to 26.6 µg of RNA/µmol total lipid. Syringe pumps (Harvard apparatus, MA, USA or YMC Co., Ltd., Kyoto, Japan) were used to control the flow rate. The resulting LNP solution was then dialyzed for 2 h or more at 4 ◦C against 20 mM MES buffer (pH 6.0), followed by phosphate-buffered saline without Ca2+ and Mg2+ (PBS(-)) using Spectra/Por 4 dialysis membranes (molecular weight cut-off 12,000–14,000 Da, Spectrum Laboratories, Rancho Dominguez, CA, USA) to remove ethanol and adjust pH to neutral. For screening A and B, 0.1 mol% of DiO was added to the lipid solution, and a mixture of siPLK1 and mNluc at 19:1 (weight ratio) was used.
