Preparation of LNPs

The LNPs were prepared by a tertiary butanol (t-BuOH) dilution procedure, as previously reported.33,37,38 The lipids, which include 1050 nmol of YSK lipids (YSK05 or YSK12-C4 or a mixture of both), 450 nmol of cholesterol, and 30 nmol of mPEG_2k-DMG – which represent a molar ratio of 70:30:2, respectively – were first dissolved in 400 µL of 90% (v/v) t-BuOH. When a fluorescent material (DiI or DiD) was incorporated into the LNPs, it was added at a concentration of 0.5–1 mol% or 0.15 mol% (of the total lipid) to the lipid solution. A 200 µL portion of an aqueous solution containing 40 µg siRNA was then added gradually to the lipid solution under vigorous mixing, producing an siRNA/lipid ratio of 0.042 (wt/wt). The siRNA–lipid solution was then gradually added to 2 mL of 20 mM citrate buffer (pH 4.0) under vigorous mixing to facilitate the precipitation and formation of the LNPs. This yields a final t-BuOH concentration of 60% (v/v). Finally, ultrafiltration using Amicon ultracentrifugal tubes (Merck Millipore Ltd, Darmstadt, Germany) was performed to remove the t-BuOH, replace the external buffer with PBS (−) (pH 7.40), and concentrate the LNPs. Centrifugation was performed at 1,000×g for 11–18 minutes at RT.