Abstract 2359 Investigation of cytocompatibility and cellular internalization of novel, enzyme-cleavable peptides

Materials and Methods: ES-2 and OVCAR-3 ovarian cancer cells were cultured in 96-well plates at 5k cells and 10k cells, respectively for cytotoxicity and internalization studies. ES-2 and OVCAR-3 cells were treated with peptides (VA-LD and GF-LD) complexed with non-targeting siRNA (siNT) at various N:P ratios for 24 and 48 hrs. To investigate the cytotoxic effects due to cathepsin B-inhibition (CTSB), ES-2 cells were treated with two CTSB inhibitors, E64D and Z-FA-FMK for 24 hours, then subsequently treated with peptide-siRNA complexes for an additional 48 hours. Cytotoxicity was measured via an MTS assay. Cellular uptake of peptides at various N:P ratios in ES-2 and OVCAR-3 cells was visualized via fluorescence microscopy at 4h using fluorescently labeled siNT and quantified using flow cytometry. Results and Conclusion: Both VA-LD and GF-LD peptides were cytocompatible with ES-2 cells and OVCAR-3 cells at N:P ratios of 20:1-40:1. However, at higher N:P ratios, cellular death was observed. Following CTSB inhibition, VA-LD and GF-LD peptides were cytocompatible at higher ratios. At N:P ratios of 20:1-40:1, cellular uptake of siRNA was visualized in ES-2 and OVCAR-3 cells at 4h. Flow cytometry confirmed over 80% internalization of both peptides in both ES-2 and OVCAR-3 cells. As the N:P ratio increased, internalization of siRNA also increased. This work demonstrated that both VA-LD and GF-LD peptides are cytocompatible at low N:P ratios, and cytocompatible at high N:P ratios after CTSB inhibition. Additionally, both peptides can effectively deliver siRNA into two ovarian cancer cells lines. Overall, VA-LD and GF-LD peptides could be further used with targeting siRNA to knock down specific oncogenic genes. It was funded in-part by Materials Assembly and Design Excellence in South Carolina under National Science Foundation Award #OIA-1655740 and SC BIOCRAFT facilities supported by the National Institute of General Medical Sciences of NIH under award number P30 GM131959, Clemson's Call Me Doctor Program (KLA) and Clemson's Creative Inquiry.