posted on 2020-09-04, 08:44authored byXinyang Zhou, Yufei Pan, Zheng Li, Huantong Li, Jing Wu, Yuan Ma, Zhu Guan, Zhenjun Yang
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.