siRNA Packaged with Neutral Cytidinyl/Cationic/PEG Lipids for Enhanced Antitumor Efficiency and Safety In Vitro and In Vivo.

The mutant BRAF gene is widely expressed in melanoma, and it acts as a suitable antitumor target. Small interference RNA (siRNA)-based therapy for BRAFV600E mRNA is, therefore, a path for melanoma clinical treatment owing to its high specificity. Although the U.S. Food and Drug Administration (FDA) approved the liver-target siRNA therapies, obstacles to siRNA tumor-targeted delivery still exist. Thus, an efficient tumor delivery system is an emergency. Here, we first report that the neutral cytidinyl lipid 2-(4-amino-2-oxopyrimidin-1-yl)-N-(2,3-dioleoyl-oxypropyl)acetamide (DNCA) could encapsulate and transfer siRNA into the cytoplasm to induce gene silencing. Also, we sought the best formulation of DNCA/dioleoyl-3,3'-disulfanediylbis-[2-(2,6-diaminohexanamido)]propanoate (CLD)/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (PEG2000-DSPE) for delivering siMB3, a siRNA for specific silencing of BRAFV600E mRNA. In the optimized formulation, the molar ratio of DNCA/CLD to a single nucleotide in siMB3 was 0.5/0.75/1 (the N/P ratio was about 3/1). Thanks to multiple forces including π-stacking, H-bonding, and electrostatic force between siRNA and lipids, the siRNA dose for effective gene silencing (85% knockdown) was reduced to 10 nM in vitro. Moreover, the siRNA lipoplexes with an additional 0.7% PEG-DSPE had a slightly negative charge and entered the cell mainly by caveolae-mediated endocytosis and macropinocytosis, avoiding degradation in the lysosome. These siRNA lipoplexes administrated through the tail vein also showed superior antitumor activity, with quite good safety and tissue distribution in vivo.