Poster T2 - Novel HER2 Targeting Tandem Peptides for delivery of CD44 siRNA into HER2+ Breast Cancer Cells in vitro.

Introduction: HER2, human epidermal growth factor receptor two, is over expressed in 20-25% of invasive breast cancers [1,2]. HER2+ breast cancer has a worse prognosis compared to other types of breast cancer, noted with higher mortality in early stages and increased relapse rates. Current treatment methods, such as surgery, chemotherapy, and hormone therapy, are often ineffective or incompatible with patients, leading to a need for new treatment options. In this study, we investigated a novel treatment for HER2+ breast cancer using peptide-mediated delivery of CD44-specifc siRNA. We characterized two novel tandem peptides consisting of a HER2 targeting peptide, P25 or P51, and the fusogenic peptide, DIV3W. By linking these two functional peptides, we aim to show that our tandem peptides can target the HER2 receptor and successfully deliver siCD44 to initiate the silencing of CD44 on the genomic and protein level in HER2+ breast cancer cells. Characterization assays were performed to determine which tandem peptide possessed more optimal qualities as a carrier for siRNA. We observed that the tandem peptide, P25-DIV3W, protects siRNA from degradation, is cytocompatible, and promotes efficient internalization into HER2+ cell lines. We additionally showed that P25-DIV3W loaded with siCD44 initiates the knockdown of CD44 and promotes cell death in HER2+ cell lines while not affecting the viability of normal breast epithelial cells in low concentrations.

Materials and

Methods: Two tandem fusogenic peptides, P25-DIV3W and P51-DIV3W were manufactured by LifeTein Inc. These peptides were tested in four cell lines: three HER2+ breast cancer cell lines (BT474, SKBR3, and MMTV-neu) and one normal breast epithelial line (MCF10A) to serve as a control. Tandem peptides were electrostatically complexed with non-targeting siRNAs at different N:P ratios to determine the minimum binding ratios needed to form nanoparticles. Different ratios of peptide-siRNA complexes were tested to determine the minimum molar ratio at which the peptides would protect siRNA from degradation. MTS assays were performed to determine cytotoxicity levels at different peptide concentrations. Dynamic light scattering (DLS) assay was performed to determine peptide-siRNA nanoparticle size and zeta potential. Fluorescent microscopy was used to determine the targeting of the peptide to HER2+ cells compared to cells not expressing HER2. Western blotting was used in a knockdown study to observe changes in protein expression in HER2+ cell lines after being treated with P25-DIV3W complexed with siNT or siCD44.

Results, Conclusions, and Discussions: Results show that both peptides target HER2+ breast cancer and are compatible carriers of siRNA, protecting the siRNA well within a cell-like environment. However, from MTS studies, P25-DIV3W is less cytotoxic to the cell lines than P51-DIV3W, which shows higher cytotoxicity at high molar ratios. Both peptides show the ability to protect siRNA from degradation via RNase A, with P25-DIV3W being the better of the two peptides. Through cellular uptake and knockdown studies, the P25-DIV3W peptide has demonstrated the ability to target HER2+ cancerous cell lines and not affect HER2 normal cells. Uptake images show around 80% uptake in the SKBR3 cell line in the 20:1 and 30:1 peptide concentrations, illustrating that the peptide accurately targets HER2+ cells and that siRNA is internalized into the cell (Figure 1). This data suggests not only that the peptide nanoparticles will not affect normal breast epithelial cells, but will also induce mRNA knockdown of the target oncogene when complexed with siCD44. P25-DIV3W at the 30:1 concentration is the optimal candidate for moving forward to in vivo studies to show the effects of CD44 knockdown in xenograft HER2+ tumors.

Acknowledgements (Optional): This research was supported in part by the Dabo Swinney All-In Foundation as well as Materials Assembly and Design Excellence in South Carolina (MADE in SC) under the National Science Foundation EPSCoR Program under NSF Award #OIA-1655740. Data presented in this material was partially collected using SC BioCRAFT facilities supported by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under award number P30 GM131959.