LNP formulation and characterization

For the preparation of LNPs, the desired lipids and siRNA were dissolved in 95% ethanol and 50 mM citrate buffer (pH = 5.0), respectively. The ethanol solution of lipids contained DoGo-derived ionizable lipid (DMB-, or DEP-, or DME-, or DEE-DoGo1), the helper lipid (DSPC), cholesterol, and DGM-PEG₂₀₀₀ in a molar ratio of 50:10:38.5:1.5. Equal volumes of lipid solution and siRNA solution (50 nM) were mixed and diluted into an equal volume of 50 mM citrate buffer (pH = 6.0). The mixture was repeatedly extruded through a 30-μm membrane (Avanti Polar Lipids, Inc., AL, USA). Then, the lipid complexes were dialyzed in a 10 KMWCO slide-A-Lyzer mini dialysis device (Thermo Fisher Scientific, MA, USA) overnight against 1x DPBS (pH = 7.4) at 4°C to remove ethanol.

The morphology of the LNPs was characterized by TEM (FEI, 80 kV, USA). The mean diameter and zeta potentials of the LNPs after dialysis was determined by dynamic light scattering (Malvern Zetasizer Nano instrument, Malvern, UK). Lipid concentrations and encapsulation efficiency (EE) were assessed by measuring the concentration of unencapsulated siRNA in LNPs. Briefly, the LNPs containing FAM-labeled siRNA after dialysis were centrifuged at 15,000 rpm for 10 min, and the supernatant fluorescence intensity (Ex/Em = 480/520 nm) was measured using FilterMax F5 Multi-Mode Microplate Reader (Molecular devices, CA, USA). The calculation formula was EE (%) = (F₀ − F₁)/F₀ × 100%, where F₀ and F₁ were the fluorescence intensity of siRNA used for LNP formulations and in the supernatant, respectively. The pKa of each ionizable lipid formulation was determined by TNS binding assay described previously.⁵⁴

The stability of lipid in full medium was investigated as follows: DEP-DoGo1 was dissolved in ethanol to a final concentration of 4 mg/mL; 5 μL of DEP-DoGo1 ethanol solution was added to 20 μL full medium (DMEM medium containing 10% fetal bovine serum) and mixed thoroughly. After incubating the mixture at room temperature for 0, 1, 2, and 3 days, the concentration of DEP-DoGo1 in each incubation was measured by TLC using dichloromethane (DCM) and methanol (10:1 volume ratio) as the eluent. The lipid spots on the TLC plate were visualized using iodine vapor. The ethanol solution of DEP-DoGo1 and the full medium were used as control. and the results were quantified by ImageJ software.

To prepare Ad LNPs, Ad1-DoGo1 or Ad4-DoGo4 was mixed with DEP-DoGo1, cholesterol, DSPC, and DMG-PEG₂₀₀₀ at the required molar ratio (Table S2), respectively, and dissolved in 95% ethanol. Then, the ethanol solution and critic acid (pH = 5.0) solution of siRNA (50 nM) were mixed thoroughly at equal volumes, then diluted by an equal volume of 50 mM citrate buffer (pH = 6.0). The mixture was repeatedly extruded through a 30-μm membrane (Avanti Polar Lipids, Inc., AL, USA). The lipid complexes were dialyzed in 10 KMWCO slide-A-Lyzer mini dialysis device (Thermo Fisher Scientific, MA, USA) overnight against 1xDPBS (pH = 7.4) in 4°C.