Dual peptide-mediated targeted delivery of bioactive siRNAs to oral cancer cells in vivo

Abstract

Objectives: Despite significant advances in cancer treatment, the prognosis for oral cancer remains poor in comparison to other cancer types, including breast, skin, and prostate. As a result, more effective therapeutic modalities are needed for the treatment of oral cancer. Consequently, in the present study, we examined the feasibility of using a dual peptide carrier approach, combining an epidermal growth factor receptor (EGFR)-targeting peptide with an endosome-disruptive peptide, to mediate targeted delivery of small interfering RNAs (siRNAs) into EGFR-overexpressing oral cancer cells and induce silencing of the targeted oncogene, cancerous inhibitor of protein phosphatase 2A (CIP2A).

Materials and methods: Fluorescence microscopy, real-time PCR, Western blot analysis, and in vivo bioimaging of mice containing orthotopic xenograft tumors were used to examine the ability of the dual peptide carrier to mediate specific delivery of bioactive siRNAs into EGFR-overexpressing oral cancer cells/tissues.

Results: Co-complexation of the EGFR-targeting peptide, GE11R9, with the endosome-disruptive 599 peptide facilitated the specific uptake of siRNAs into oral cancer cells overexpressing EGFR in vitro with optimal gene silencing observed at a 60:30:1 (GE11R9:599:siRNA) molar ratio. Furthermore, when administered systemically to mice bearing xenograft oral tumors, this dual peptide complex mediated increased targeted delivery of siRNAs into tumor tissues in comparison to the 599 peptide alone and significantly enhanced CIP2A silencing.

Conclusion: Herein we provide the first report demonstrating the clinical potential of a dual peptide strategy for siRNA-based therapeutics by synergistically mediating the effective targeting and delivery of bioactive siRNAs into EGFR-overexpressing oral cancer cells.

Keywords: CIP2A; Cell-penetrating; EGFR; Endosome-disruptive; Homing/targeting; OSCC; Oral cancer; Peptide; RNAi; siRNA.

Fig. 1

The GE11R9 peptide targets delivery of siRNAs to cells with high expression of EGFR, but is not capable of delivering bioactive siRNAs. (A) Fluorescence microscopy analysis of targeted siRNA delivery following co-culture of high EGFR-expressing CAL 27 and low EGFR-expressing SCC-15 oral cancer cells incubated for 4 hours with DY547-conjugated siCIP2A (DY547-siCIP2A, red) in complex with GE11R9 peptide at 100:1 or 60:1 (GE11R9:DY547-siCIP2A) molar ratios. Of note, to distinguish between the two cell lines in co-culture, cells were stained for vimentin (green, top panels), which was found to be differentially expressed, with CAL 27 cells being vimentin negative and SCC-15 cells being vimentin positive. Nuclei (blue) were counterstained with DAPI. The bottom panels are identical to the top panels, except that the vimentin fluorescent signal was removed in the bottom panels to better visualize the degree of fluorescent siRNA signal in SCC-15 cells, which are indicated by arrowheads. Scale bar: 100 μm. (B) Western blot analysis of EGFR and vimentin protein expression levels in CAL 27 and SCC-15 cells. GAPDH protein levels were monitored to ensure equal loading of samples. (C) Western blot analysis of CIP2A protein expression levels in CAL 27 cells performed 48 hours post-treatment with GE11R9 peptide complexed to either a control non-targeting siRNA (siNT) or siCIP2A at a 60:1 or 100:1 (GE11R9:siRNA) molar ratio compared to 599 peptide at a 50:1 (599:siRNA) molar ratio or untreated cells. β-Actin protein levels were monitored to ensure equal loading of samples. (D) Indirect immunofluorescence microscopy analysis of endosomal co-localization following transfection of CAL 27 cells with DY547-siCIP2A (red) complexed with GE11R9 at 100:1 or 60:1 (GE11R9:DY547-siCIP2A) molar ratios or 599 peptide at a 50:1 (599:DY547-siCIP2A) molar ratio for 4 hours. In the top panels, cells were stained for endosomes (green) and nuclei (blue) were counterstained with DAPI. The co-localization of DY547-siCIP2A with endosomes (white; bottom panels) was determined using the ImageJ [44] Colocalization plug-in (National Institutes of Health, Bethesda, MD; freely available at imagej.nih.gov/ij/). Scale bar: 50 μm.

Fig. 2

The dual peptide complex retains the ability to specifically target siRNAs to cells overexpressing EGFR. Fluorescence microscopy analysis of the co-culture of CAL 27 (high EGFR, vimentin negative) and SCC-15 (low EGFR, vimentin positive) oral cancer cells incubated for 4 hours with DY547-siCIP2A (red) in complex with GE11R9 and/or 599 peptides at various molar ratios (GE11R9:599:DY547-siCIP2A). Cells were stained for vimentin (green) and nuclei (blue) were counterstained with DAPI. Scale bar: 100 μm.

Fig. 3

The dual peptide complex mediates delivery of bioactive siRNAs into oral cancer cells. (A) Western blot analyses of CIP2A protein expression levels in CAL 27 cells 48 hours post-treatment with siNT or siCIP2A in complex with GE11R9 and/or 599 peptides at various GE11R9:599:siRNA molar ratios. β-Actin protein levels were monitored to ensure equal loading of samples. (B) Real-time PCR analysis of CIP2A mRNA levels in CAL 27 cells 48 hours post-treatment with siNT or siCIP2A in complex with either the dual peptide at various GE11R9:599:siRNA molar ratios or the 599 peptide alone. The CIP2A mRNA levels were normalized to 18S rRNA. Data are mean ± SEM of three independent experiments performed in triplicate, where *P<0.05, ***P<0.001, ****P<0.0001, and P≥0.05 is not significant (ns) compared to siNT treated cells (Student’s t test).

Fig. 4

The dual peptide-siRNA complex displays no significant cytotoxicities at GE11R9:599:siRNA molar ratios up to 60:30:1. Assessment of long-term viability of CAL 27 cells 48 hours post-treatment with siNT in complex with the dual peptide at various GE11R9:599:siNT molar ratios. Cells were treated with siNT alone or left untreated as negative controls and untreated cells were defined as 100% viable. Data are mean ± SEM of three independent experiments performed in triplicate, where **P<0.01, ***P<0.001, and P≥0.05 is not significant (ns) compared to untreated cells (one-way ANOVA).

Fig. 5

Biochemical characterization of the dual peptide carrier at the optimized GE11R9:599:siRNA molar ratio of 60:30:1. (A) Size distribution and zeta potential of the dual peptide complexed with siCIP2A at the 60:30:1 molar ratio 20 minutes after formulation in water. (B) Agarose gel shift assay examining the ability of the dual peptide to complex free siCIP2As at the 60:30:1 (GE11R9:599:siCIP2A) molar ratio in comparison to GE11R9 or 599 at a 60:0:1 or 0:30:1 molar ratio, respectively. Non-complexed siCIP2As (0:0:1 molar ratio) and the molecular weight marker (MWM) are also shown. (C) Stability of siRNAs in complex with the dual peptide carrier was examined in the presence and absence of serum or RNase A. In particular, naked and dual peptide-complexed siCIP2As at the 60:30:1 molar ratio were incubated in the absence or presence of either 50% fetal bovine serum (FBS) or 5 μg/ml RNase A for one hour at 37ºC. The position of intact siCIP2A is indicated.

Fig. 6

The dual peptide carrier enhances delivery of bioactive siRNAs to xenograft oral cancer tumors following systemic administration. (A) Representative in vivo images of siRNA biodistribution in mice and (B) quantitative analysis of tumor fluorescence 1 to 48 hours post-treatment with Cy5.5-labeled siCIP2A (Cy5.5-siCIP2A) in complex with the dual peptide at a 60:30:1 (GE11R9:599:siRNA) molar ratio (Dual peptide+Cy5.5-siCIP2A) or the 599 peptide alone at a 0:30:1 molar ratio (599+Cy5.5-siCIP2A). Black and white image (B/W); site of tumor (arrow); Arbitrary units (A.U.). Data are mean ± SEM of four independent samples, where *P<0.05 compared to 599+Cy5.5-siCIP2A complex-treated tumors (Student’s t test). (C) Real-time PCR analysis of relative CIP2A mRNA levels in excised xenograft tumor tissues, 48 hours post-treatment with Dual peptide+Cy5.5-siCIP2A, 599+Cy5.5-siCIP2A, or dual peptide complexed to siNT (Dual peptide+siNT), normalized to untreated tissues. The CIP2A mRNA levels were normalized to 18S rRNA. Data are mean ± SEM of four independent samples performed in triplicate, where *P<0.05 and P≥0.05 is not significant (ns) compared to Dual peptide+siNT (one-way ANOVA).

Fig. 7

Model of targeted delivery and gene silencing using the dual peptide-siRNA nanocomplex. Both the GE11R9 and 599 peptides complex with siRNA using a stretch of densely packed cationic nona(D-arginine) amino acid residues, which enable complexing via electrostatic interactions. The GE11R9 peptide mediates cellular entry through receptor-mediated endocytosis initiated by binding of GE11R9 to the epidermal growth factor receptor (EGFR). Upon entering the cell, 599 mediates endosomal escape of siRNA under acidic conditions and siRNA in the cytoplasm enter the RNA-induced silencing complex (RISC), which together enable sequence-specific inhibition of gene expression.