Abstract
Although small interfering RNAs (siRNAs) have been demonstrated to specifically silence their target genes in disease models and clinical trials, in vivo siRNA delivery is still the technical bottleneck that limits their use in therapeutic applications. In this study, a bifunctional peptide named RGD10-10R was designed and tested for its ability to deliver siRNA in vitro and in vivo. Because of their electrostatic interactions with polyarginine (10R), negatively charged siRNAs were readily complexed with RGD10-10R peptides, forming spherical RGD10-10R/siRNA nanoparticles. In addition to enhancing their serum stability by preventing RNase from attacking siRNA through steric hindrance, peptide binding facilitated siRNA transfection into MDA-MB-231 cells, as demonstrated by FACS and confocal microscopy assays and by the repressed expression of target genes. When RGD10 peptide, a receptor competitor of RGD10-10R, was added to the transfection system, the cellular internalization of RGD10-10R/siRNA was significantly compromised, suggesting a mechanism of ligand/receptor interaction. Tissue distribution assays indicated that the peptide/siRNA complex preferentially accumulated in the liver and in several exocrine/endocrine glands. Furthermore, tumor-targeted delivery of siRNA was also demonstrated by in vivo imaging and cryosection assays. In summary, RGD10-10R might constitute a novel siRNA delivery tool that could potentially be applied in tumor treatment.
Highlights
Have been found to mediate adhesion between cells and the extracellular matrix (ECM) and to stimulate intracellular signaling and gene expression involved in cell growth, migration and survival[20]
Compared to RGD10-10R, compromised siRNA binding capacity was observed for polyarginine (10R) peptide without the RGD10 motif at the N terminal (Fig. 2b-right panel)
The results indicated that RGD10-10R could bind siRNA and form stable complexes in this medium, as well as in DEPC water (Fig. 2c)
Summary
Have been found to mediate adhesion between cells and the extracellular matrix (ECM) and to stimulate intracellular signaling and gene expression involved in cell growth, migration and survival[20]. RGD-based peptides have been used to facilitate siRNA transportation in vitro and in vivo, they were only incorporated into certain carriers and played a role of specific recognition[28,29,30,31,32] Such types of delivery systems are typically multicomponent, complex and difficult to develop as potential drug delivery formulations. It was previously reported that the cyclic RGD peptide exhibited considerably higher affinity toward α vβ 3 compared with the linear RGD peptide[34] In this delivery system, RGD10 was designed to recognize integrin(s) expressed on tumor cells and tumor-associated endothelial cells, and 10R was employed to bind siRNA through electrostatic interactions (Fig. 1a). We evaluated the in vivo distribution and tumor-targeting profile through fluorescent imaging and cryosection observations
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