Abstract
RNA interference has been used to dissect the importance of individual gene products in various human disease processes, including cancer. Small-interfering RNA, or siRNA, is one of the tools utilized in this regard, but specially-designed delivery agents are required to allow the siRNA to gain optimal access to the cell interior. Our laboratory has utilized two different siRNA-binding delivery peptides containing a polyarginine core, and modified by myristoylation and targeting motifs (iRGD or Lyp-1). A third peptide was designed to assist with endosomal release. Various ratios of the peptides and siRNA were combined and assayed for the ability to form stable complexes, and optimized ratios were determined. The complexes were found to form particles, with the majority having a diameter of 100–300 nm, as visualized by electron microscopy. These siRNA complexes have enhanced protection from nucleases present in serum, as compared to “naked” unprotected siRNA. The particles were internalized by the cells and could be detected in the cell cytoplasm by confocal fluorescence microscopy. In functional assays, peptide/siRNA complexes were shown to cause the knock down of corresponding targeted proteins. The peptide with the LyP-1 targeting motif was more effective at knockdown in MDA-MB-231 breast cancer cells than the peptide with the iRGD motif. Inclusion of the endosomal release peptide in the complexes greatly enhanced the peptide/siRNA effects. Peptide/siRNA complexes simultaneously targeting Stat3 and c-Myc caused a marked reduction in anchorage-independent growth, a property correlated with tumorigenicity. This study demonstrates the ability of a peptide-based siRNA-delivery system to deliver siRNA into breast cancer cells and cause both protein knockdown and suppression of the malignant phenotype. Such peptide complexes are likely to become highly useful siRNA-delivery vehicles for the characterization, and potentially for the treatment, of human cancer.
Highlights
By harnessing the power of the RNA interference (RNAi) pathway, short-interfering RNAs have become a versatile and powerful tool for identifying and characterizing the importance of specific gene(s) in disease processes such as cancer [1,2,3], and as a potential therapeutic agent for treatment of various diseases [4,5,6]
It is likely that no one delivery method will prevail for all the disease processes being currently targeted by siRNA, as the location of these disease processes varies within the body, and delivery vehicles may intentionally or unintentionally target certain tissue locations with some selectivity [12, 13], and may show selectivity towards specific cell types within tissues
We have examined the ability of synthetic peptides to complex with and deliver a siRNA cargo to human breast cancer cells
Summary
By harnessing the power of the RNA interference (RNAi) pathway, short-interfering RNAs (siRNAs) have become a versatile and powerful tool for identifying and characterizing the importance of specific gene(s) in disease processes such as cancer [1,2,3], and as a potential therapeutic agent for treatment of various diseases [4,5,6]. RNAi lends itself well to current approaches emphasizing personalized medicine combined with targeted therapy, as the length of time it takes to design, test, and produce potentially therapeutic siRNAs is much shorter than the time it takes to develop classical pharmaceuticals, and the costs are considerably less. It is likely that no one delivery method will prevail for all the disease processes being currently targeted by siRNA, as the location of these disease processes varies within the body, and delivery vehicles may intentionally or unintentionally target certain tissue locations with some selectivity [12, 13], and may show selectivity towards specific cell types within tissues
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