RESUMEN
The efficient delivery of small interfering RNAs (siRNAs) to the target cells is critical for the pharmaceutical success of RNA interference (RNAi) drugs. One of the possible strategies to improve siRNA delivery is to identify auxiliary molecules that augment their cellular uptake. Herein, we performed a chemical library screening in an effort to discover small molecules that enhance the potency of cholesterol-conjugated, cell-penetrating asymmetric siRNAs (cp-asiRNAs). Interestingly, three compounds identified from the screen share a common dihydropyridine (DHP) core and function as L-type calcium channel blockers (CCBs). Using confocal microscopy and quantitative analysis of small RNAs, we demonstrated that the L-type CCBs increased the endocytic cellular uptake of cp-asiRNAs. Furthermore, these small molecules substantially improved the potency of cp-asiRNAs, not only in vitro but also in vivo on rat skin. Collectively, our study provides an alternative pharmacological approach for the identification of small molecules that potentiate the effects of therapeutic siRNAs.
Asunto(s)
Bloqueadores de los Canales de Calcio/administración & dosificación , Bloqueadores de los Canales de Calcio/farmacocinética , Canales de Calcio Tipo L/metabolismo , Dihidropiridinas/farmacocinética , Interferencia de ARN , ARN Interferente Pequeño/farmacocinética , Animales , Biopsia , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Colesterol/química , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Dihidropiridinas/administración & dosificación , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/farmacocinética , Células HeLa , Humanos , Inyecciones Intradérmicas , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Piel/metabolismo , Piel/patología , Bibliotecas de Moléculas Pequeñas , TransfecciónRESUMEN
RNA interference (RNAi) triggering oligonucleotides in unconventional structural format can offer advantages over conventional small interfering RNA (siRNA), enhanced cellular delivery and improved target gene silencing. With this concept, we present a well-defined tripodal-interfering RNA (tiRNA) structure that can induce simultaneous silencing of multiple target genes with improved potency. The tiRNA structure, formed by the complementary association of three single-stranded RNA units, was optimized for improved gene silencing efficacy. When combined with cationic polymers such as linear polyethyleneimine (PEI), tiRNA assembled to form a stable nano-structured complex through electrostatic interactions and induced stronger RNAi response over conventional siRNA-PEI complex. In combination with a liver-targeting delivery system, tripodal nucleic acid structure demonstrated enhanced fluorescent accumulation in mouse liver compared to standard duplex nucleic acid format. Tripodal RNA structure complexed with galactose-modified PEI could generate effective RNAi-mediated gene silencing effect on experimental mice models. Our studies demonstrate that optimized tiRNA structural format with appropriate polymeric carriers have immense potential to become an RNAi-based platform suitable for multi-target gene silencing.