RESUMEN
Single-stranded siRNA (ss-siRNA) refers to the antisense strand of siRNA, which plays the role of gene silencing. Since single-stranded RNA is unstable, the present study employed a homemade neutral cytidinyl/cationic lipid delivery system and chemical modifications to improve its stability. The results showed that with the aid of mixed lipids, ss-siRNA could knock down 40% of target mRNA at 25 nM. With 2'-F/2'-OMe, phosphorothioate and 5'-terminal phosphorylation, the optimized ss-siRNA could knock down 80% of target mRNA at 25 nM. Further knocking down AGO2, the ss-siRNAs could not effectively silence target mRNAs. Analysis of the biodistribution in vivo showed that ss-siRNA was less durable than siRNA, but spread more quickly to tissues. This study provides a safe and efficient ss-siRNA delivery and modification strategy, which lays the foundation for future works.
RESUMEN
BC15-31 is a DNA aptamer that targets heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), which plays a crucial role in the process of pre-RNA maturation and is also essential for the rapid proliferation of tumor cells. In this research, we modified BC15-31 with a phosphorothioate (PS) backbone, LNA, and 2-O-MOE to enhance its stability and target affinity. In addition, a neutral cytidinyl lipid (DNCA) and a cationic lipid (CLD) were mixed to encapsulate modified aptamers with the aim of improving their cell permeability with low toxicity. Under the DNCA/CLD package, aptamers are mainly distributed in the nucleus. A modified sequence WW-24 showed an excellent selective anti-melanoma (A375 cells, â¼25 nM, 80%) activity, targeted to both hnRNP A1 and hnRNP A2/B1 found by the BLI experiment, and induced more early and late apoptosis in vitro, which also showed stronger antitumor effect and longer accumulation time in vivo. These results provide a new strategy for further clinical applications.
RESUMEN
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.