Cancer Technologies
In vitrocharacterization of an enzyme-cleavable tandem peptide for siRNA delivery to ovarian cancer cells
Ruxi Xia (she/her/hers)
Undergraduate Research Assistant
Clemson University
Clemson, South Carolina, United States
Chloe Jones (she/her/hers)
Undergraduate Research Assistant
Clemson University
Clemson , South Carolina, United States
Kharimat Lora Alatise, MS
Ph.D. Candidate
Clemson University
Central, South Carolina, United States
Angela Alexander-Bryant (she/her/hers)
Assisstant Professor
Clemson University, United States
Ovarian cancer has caused several deaths amongst women due to its late-stage diagnosis, malignant nature, and lack of effective treatment options. Overexpression of genes that promote resistance to therapeutics, metastasis, and tumorigenesis render current therapeutic options to be ineffective. However, RNA interference (RNAi) is a promising therapeutic tool that can silence such genes. RNAi can be induced via small interfering RNA (siRNA) and delivered using peptide nanocarriers. This work assesses the ability of two targeted, stimuli-sensitive peptides, to deliver siRNA to ovarian cancer cells in vitro. Each peptide is comprised of a targeting, enzyme cleavable, and fusogenic region; however, they differ in sequence for the enzyme-cleavable region to determine which peptide will perform best in delivering siRNA. In vitro studies were conducted to observe their toxicity, internalization into ovarian cancer cells, ability to mediate and endosomal escape and gene knockdown.
Human ovarian cancer ES-2 and OVCAR-3 cells were cultured in 96-well plates at 5,000 cells and 20,000 cells, respectively. ES-2 and OVCAR-3 cells were treated with peptides complexed with non-targeting siRNA (siNT) at various N:P ratios for 24 and 48 hrs. Cytotoxicity was then assessed via an MTS assay. For cellular uptake of peptides, ES-2 cells were seeded with a density of 10,000 cells/well and were treated with peptides at various N:P ratios in ovarian ES-2 cells was visualized via fluorescence microscopy at 4h using fluorescently labeled siNT. To visualize endosomal escape, ES-2 cells were cultured in a 96 well plate and seeded at 8,000 cells/well. ES-2 cells were treated with peptides complexed with CY5 labeled siNT and incubated for either 30 minutes, 2 hours, and 4 hours. The cells were then stained with Hoechst 33342, and the early endosome antigen 1 antibody then imaged via fluorescence microscopy at each time point. Finally, ES-2 cells were cultured in a 24 well plate seeded at 20,000 cells respectively, then treated at various N:P ratios of peptides using siNT or siCSNK2A1 (target gene) for 48 hours. In this study, a non-cleavable peptide and fusogenic peptide were used as controls.
Both cleavable peptides are biocompatible with ovarian cancer cells at low N:P ratios and can deliver siRNA to two ovarian cancer cell lines at 4 hours. Additionally, each cleavable peptide can mediate endosomal escape, at as early as 2 hours, regardless of the N:P ratio. Therefore, we expect to see significant mRNA reduction of our target gene as well as protein silencing. Future studies will examine protein silencing via western blotting.
This research was funded in-part by Materials Assembly and Design Excellence in South Carolina (MADE in SC) under National Science Foundation (NSF) Award #OIA-1655740 (AAB) and the NSF CAREER Award #2046694 (AAB). Data presented in this publication 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 (AAB). Any opinions, findings, conclusions, or recommendations expressed in the material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation and National Institutes of Health. Support was also provided by Clemson’s Call Me Doctor Fellowship (KLA) and the College of Engineering, Computing, and Applied Sciences through their Undergraduate Opportunity research grant program, by the Clemson University Honors College, and by Clemson Creative Inquiry (RX).