Small interfering RNA (siRNA) therapeutics hold great potential for treating genetic disorders and cancer, yet their clinical translation remains constrained by the challenge of efficient delivery. This study systematically investigates the effect of the ratio between cationic and helper lipids in the formulation of 2X3-DOPE-based liposomes, including formulations equipped with PEG-shielding lipoconjugates differing in structure and PEG (polyethylene glycol) length on the efficiency of siRNA accumulation and gene silencing. Changing the 2X3-to-DOPE cationic-to-helper lipid molar ratio from 1:1 to 1:2 differentially affects the properties of formulations across N/P ratios, but has little effect on the transfection efficiency in cell culture. Pharmacokinetic analysis in mice revealed that siRNA complexed with PEGylated P2000 1:1 liposomes achieved the highest plasma concentration (14.23 pmol/mL at 15 min), representing a 41-fold increase over non-PEGylated formulation, but exhibited only moderate hepatic Ttr gene silencing (35.5%). Remarkably, 2X3 1:1 liposomes demonstrated exceptional silencing efficacy (93.9% knockdown), while increasing helper lipid content reduced the activity of complexes with 2X3 1:2 liposomes (80.2% decrease in Ttr gene knockdown). The study shows that siRNA therapeutics require formulation-specific balance between systemic stability, hepatocyte uptake, endosomal escape, and RISC loading and highlighted the indispensable role of cationic-to-helper lipid ratio optimization to maximize bioactivity without sacrificing PEG-mediated protection.
Gladkikh et al. (Thu,) studied this question.