Research Article: Delivery of gene targeting siRNAs to breast cancer cells using a multifunctional peptide complex that promotes both targeted delivery and endosomal release

Date Published: June 30, 2017

Publisher: Public Library of Science

Author(s): Jeffrey D. Bjorge, Andy Pang, Donald J. Fujita, Maxim Antopolsky.


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.

Partial Text

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 [1–3], and also as a potential therapeutic agent for treatment of various diseases [4–6]. In particular, 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. Importantly, almost any gene or combination of genes can be readily targeted for suppression. Despite its promise, one major obstacle that investigators have had to overcome when developing siRNA-based pharmaceuticals has been the delivery of these highly charged molecules to the targeted cells. Many different methods of delivery are currently under development, each with its own set of advantages [7–11]. 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 also show selectivity towards specific cell types within tissues.

We synthesized several peptides that would form multifunctional complexes with properties that would enable the delivery of siRNA into breast cancer cells. These properties included the ability to form small particles in aqueous solutions, to non-covalently bind to and act as a carrier for siRNA, to bind to and be internalized by human breast cancer cells, and finally, to release the siRNA inside the cell to allow suppression of target genes. The domain structure of these peptides can be summarized in Fig 1. Two siRNA-binding peptides, myr-R9-LyP-1 and myr-R9-iRGD were synthesized. They consist of a hydrophobic myristoylated amino-terminus (myr) that facilitates the formation of small particles and assists with the transiting of the particles across cellular membranes. Next, there is a positively-charged arginine-rich segment which allows binding via electrostatic interaction with the negatively charged siRNA and with the plasma membrane. Finally, there is a tumor targeting sequence (either LyP-1 or iRGD) that targets the peptides to cells possessing complementary binding sites. LyP-1 and iRGD were originally identified using phage display library screening [27, 28] and have been shown to target a variety of tumor types [28–30]. A third peptide, an endosomal-release peptide (E9), was synthesized to contain a domain capable of mediating intracellular endosomal release and nine negatively-charged glutamic acid residues, which facilitate the electrostatic interaction with the siRNA-binding peptides.

In this study, we have examined the ability of synthetic peptides to complex with and deliver a siRNA cargo to human breast cancer cells. We have designed these peptides utilizing currently available information and have utilized and combined peptide sequences/motifs and modifications from several sources that we felt would best facilitate the many steps in the delivery process. These steps include the binding of siRNA, the safe transport of the peptide/siRNA complex to cells, the uptake of the peptide/siRNA complex into the cytoplasm of the cells, and finally the release of the siRNA from the complex and incorporation into the RISC silencing complex.




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