Investigating the enzymatic processing and gene knockdown of micelle-conjugated siRNA against glioblastoma

摘要

Introduction: Glioblastoma (GBM), a type of brain cancer, is one of the deadliest cancers and represents a significant clinical challenge. It is hypothesized that GBM cancer stem cells (GSCs) are the major cell type responsible for glioblastoma invasion and recurrence. Small interfering ribonucleic acids (siRNAs) can be engineered to target specific proteins responsible for cancer cell proliferation and invasion.However, these nucleic acids are susceptible to cleavage in vivo. Polymeric nanomicelles are promising vehicles for the delivery of genetic therapeutics to cancerous tissue, stabilizing the nucleic acids with respect to cleavage and enabling targeted delivery strategies. As an additional stabilization strategy, Damha and coworkers have synthesized nucleic acids that substitute 2'-ribose for 2'-fluoro-ribose (FRNA), 2'-fluoro-β-D-arabinose (FANA) or β-D-arabinose (ANA) in the oligonucleotide chain. These modifications increase the intracellular half-life of the resulting siRNA. siRNAs that are synthesized as Dicer substrates (an important enzyme for siRNA cell trafficking) show enhanced efficacy. The Dicer processing of siRNA-polymer systems has been examined for poly(ethylene glycol) and lipid conjugates, but these studies are scarce. Importantly, the Dicer processing of fluoro-modified siRNAs has not been directly investigated. In this work, the Dicer processing of fluoro-modified, micelle-conjugated siRNA will be presented. The gene knockdown in GSCs is also investigated using these dicer-substrate siRNAs. A gene associated with the GSC invasion, resistance, and sternness was chosen as the genetic target in a series of primary GSC lines. Materials and Methods: Oligonucleoticles are synthesized using standard phosphoramidite chemistry. Micelles are synthesized as previously described and the siRNA-micelle conjugation is carried out by a Huisgen dick type reaction between the DBCO-functionalized oligonucleotide and the azide-functionalized micelle. Dicer enzyme kit was used as purchased from Genlantis. The electrophoretic mobility of oligonucleoticles and digested products were analyzed using native polyacrylamide electrophoresis. Individual gel lanes were quantified using density measurements obtained with Image J software. Gene knockdown studies were performed using Lipofectamine 2000 (purchased from Invitrogen) in a 6-well format. Results and Discussion: siRNA duplexes were designed with varying substitution patterns (unmodified, partly modified, and fully modified). Dicer processing was conducted on each siRNA duplex, both unconjugated and conjugated to the micelle, and digested products analyzed by native PAGE. Significant processing was observed in all cases, though the unmodified strands were observed to undergo the most enzyme cleavage. Significant gene knockdown was observed when transfecting the siRNA into glioblastoma cells, both unconjugated and conjugated to the micelle. Conclusions: The results of this study indicate that Dicer is still able to effectively process modified siRNA, even with the duplexes conjugated to a micelle. In addition, the siRNA sequences developed here were able to regulate gene expression in glioblastoma stem cells. Looking ahead, these results are a promising indicator of siRNA potency and their incorporation into a targeted nanomicelle delivery system for GBM therapy is currently under investigation.