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

<div><p>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.</p></div

Confocal microscopy of MDA-MB-231 cells incubated with myr-R9-LyP-1/siRNA complexes.

<p>Peptide/siRNA complexes were formed as described in the material and methods using fluorescently-labelled siRNA-binding peptide. The complexes were incubated on MDA-MB-231 cells for 18 hours, and the cells were then live-mounted onto glass slides. The slides were viewed by confocal microscopy, and serial sections through one field of cells are shown (panels 1–4). The fluorescently-labeled siRNA-binding peptide is shown in yellow, and the plasma membrane is shown in red (Cell Mask plasma membrane stain). The results presented are representative of three independent experiments.</p

Stat3 knockdown by siRNA delivered to the MDA-MB-231 cells using myr-R9-LyP-1 or myr-R9-iRGD.

<p>Peptide/siRNA complexes were formed with optimized ratios of siRNA binding peptide, siRNA, and endosomal release peptide, and incubated on the cells overnight. After 48 hours, cell extracts from the MDA-MB-231 cells were examined for Stat3 protein expression by Western blotting. A representative visualized Stat3 blot is shown along with a bar graph displaying the quantitated results derived from three independent experiments (means +/- SEM; results normalized to 100% relative to the mean of one triplicate set of untreated samples). Statistical analysis consisted of Analysis of Variance and Tukey's pairwise comparison. Significant differences are indicated (* p < 0.001 and ** p < .01).</p

c-Myc knockdown by siRNA delivered to the MDA-MB-231 cells using myr-R9-LyP-1 or myr-R9-iRGD.

<p>Peptide/siRNA complexes were formed with optimized ratios of siRNA binding peptide, siRNA, and endosomal release peptide, and incubated on the cells overnight. After 48 hours, cell extracts from the MDA-MB-231 cells were examined for c-Myc protein expression by Western blotting. A representative visualized c-Myc blot is shown along with a bar graph displaying the quantitated results derived from three independent experiments. (means +/- SEM; results normalized to 100% relative to the mean of one triplicate set of untreated samples). Statistical analysis consisted of Analysis of Variance and Tukey's pairwise comparison. Significant differences are indicated (* p < 0.001 and ** p < .01).</p

Inhibition of soft agar colony formation by the combination of Stat3 and c-Myc siRNA complexed with myr-R9-LyP-1 and E9 peptides.

<p>MDA-MB-231 cells were either left untreated or incubated with control siRNA or a combination of Stat3 and c-Myc siRNAs complexed with either Lf-2000 or myr-R9-LyP-1 and E9 peptides. After 24 hours, the cells were detached with trypsin and replated in soft agar as described in the materials and methods. After 2 weeks, cell colonies larger than 50 cells were quantitated and the results shown. The height of each bar is calculated from the mean of triplicate measurements +/- 1 S.D, and the data analyzed by Analysis of Variance and Tukey's pairwise comparison. Significant differences are indicated (* and ** p < .001). A representative photomicrograph is shown from each condition.</p

Binding to the targeting peptide protects the siRNA from serum-mediated degradation.

<p>Control non-targeting siRNA was either untreated (No peptide) or incubated with myr-R9-LyP-1 peptide (+ peptide) and E9 to form peptide/siRNA complexes. The unbound or bound siRNA were then incubated in the presence of bovine serum for the indicated lengths of time and then separated on a 7M urea gel. The siRNA bands were visualized by staining with Sybr Green II (upper panel). The full-length 21 nucleotide band was quantitated using ImageQuant software (GE) and the results shown in the lower panel. The results presented are representative of three independent experiments.</p

Visualization of the peptide/siRNA peptide complexes by electron microscopy.

<p>Peptide/siRNA complexes were stained with uranyl acetate and visualized on a transmission electron microscope. Numerous stained spherical particles were observed. Two representative fields are shown, one at 1500X (top) and 5000X (bottom) magnification. The particles from two separate fields at 1500X and 1200X magnification were measured (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180578#pone.0180578.s001" target="_blank">S1 Fig</a>) and the particle distribution is shown in the graph.</p

Optimization of the binding of peptide E9 to the siRNA-binding peptides.

<p>Various amounts of peptide E9 were added to the siRNA-binding peptides myr-R9-LyP-1 and myr-R9-iRGD, followed by 0.10 nmoles of siRNA. Following 15 minutes of incubation, the free and bound siRNA was separated on a 2.5% non-denaturing agarose gel. The gel was stained with Sybr Green II RNA stain, and the resulting fluorescent bands were visualized with a Fuji LAS4000 Imager. The results presented are representative of three independent experiments.</p

Src siRNA causes apoptosis of MDA-MB-435S cells.

<p>MDA-MB-435S cells were transfected with siRNA or treated with 1 uM staurosporine. 48 hours post-treatment, the cells were collected and assayed for apoptosis using the TUNEL assay. Results are expressed with log(fluorescence) on the horizontal axis and cell counts on the vertical axis and is representative of triplicate experiments.</p

Injection of siRNA/oligofectamine complexes into mouse tumors inhibits tumor growth.

<p>MDA-MB-435S cells were implanted in the mammary fat pad of SCID/NOD mice. Two days post-implantation, the mice were divided into 3 groups (one group of 10 control siRNA-injected mice and two groups of 5 targeted siRNA mice) and the groups received twice weekly injections of either control, Src+STAT3, or Src+STAT3+Myc siRNA into the site of the cell implantation/tumor. 47 days post-implantation, 5 mice in the control group were switched to Src+STAT3 siRNA injections for the remainder of the experiment (Control, then Src+STAT3 group). Tumor growth was monitored and the results are shown (<b>A</b>), with each treatment group shown in a single panel, and individual mouse tumors represented by connected data points. 126 days post-implantation, the primary tumor(s) were excised (<b>B</b>) and weighed. The tumor weights for each mouse (data points) and the average tumor weight for each group (horizontal bar) is shown (<b>C</b>). Tumor weight data was analyzed by Analysis of Variance and Tukey's pairwise comparison, with significant differences between the control group and two of the treatment groups noted ( *p<0.05).</p