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Drug Delivery

Drug Delivery - Poster Session C

Poster J3 - Investigating the GE11-599 Tandem Peptide for Delivery of siRNA to treat Glioblastoma

Friday, October 25, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Glioblastoma (GBM) is the most prevalent primary brain tumor of the central nervous system. Current treatments include surgical resection followed by adjuvant chemotherapy and/or radiotherapy. Despite current treatment options, the prognosis remains poor. RNA interference (RNAi) is a promising therapeutic that has proven effective in treating various diseases, including GBM. The mechanism for RNAi is to target specific oncogenes that drive cancer progression, utilizing short interfering RNA (siRNA) to target a specific messenger RNA (mRNA). By nature, siRNA is presented with delivery challenges as it's easily degradable; therefore, a delivery system is needed. Peptide-based delivery systems have been shown to enhance delivery and cancer targetability.1 Peptides are typically around 50 or fewer amino acids in length and are easily tunable, biocompatible, and affordable drug delivery systems. This study aims to develop a multifunctional peptide, termed tandem peptide, to overcome numerous delivery barriers associated with RNA delivery. The GE11 peptide targets the epidermal growth factor receptor (EGFR), which is overexpressed in GBM cells. The 599 peptide facilitates endosomal escape. This study uses a GE11-599 tandem peptide to bind to EGFR, release a bioactive siRNA into the cytoplasm, and then silence the target gene STAT3.

Materials and

Methods:  The GE11-599 tandem peptide was synthesized, purified, and analyzed by Lifetein LLC. The human glioblastoma cell line used in the following experiments was a U118 cell line obtained from the American Type Culture Collection (ATCC). The tandem peptide underwent a gel shift assay at various N:P (peptide:siRNA) ratios to determine the minimum ratio needed to complex free siRNA. To evaluate the ability of the peptide to protect siRNA from degradation, GE11-599-siRNA complexes were left within water or degradative environments, 50% FBS or RNase A, and ran through gel electrophoresis to image intact white bands. The viability of U118 cells following peptide-siRNA treatment was assessed using an MTS assay in a 96-well plate to investigate the cytocompatibility of complexes. Next, qRT-PCR was conducted to evaluate STAT3 gene silencing on the mRNA level following treatment with GE11-599-siSTAT3. To assess cell migration following peptide-mediated STAT3 silencing, a wound-healing assay was performed by scratching a gap in the cellular monolayer and monitoring over 48 hours. Migration was quantified with ImageJ.

Results, Conclusions, and Discussions: A gel shift assay of the GE11-599 tandem peptide shows that the tandem peptide electrostatically complexes with siRNA up to a 20:1 N:P ratio. A gel shift protection assay was conducted to mimic physiological conditions and assess the peptides' ability to protect the siRNA. Results show that the tandem peptide could protect the siRNA in two environments: physiological relevant, 50% fetal bovine serum (FBS), and a harsher degradation environment, RNAse A. The white intact banding shows the peptides' ability to withstand these conditions. Next, an MTS viability assay was conducted to evaluate the cytocompatibility of peptide complexes, showing minimal cytotoxicity in U118 GBM cells. Endosomal escape assay was conducted through immunofluorescent imaging where the nuclei were stained blue with DAPI, siRNA was stained red, and endosomes were stained green. Separate localization of green and red suggests that the tandem peptide facilitated the endosomal escape of siRNA. qPCR was used to compare STAT3 mRNA expression in U118 cells after peptide-siSTAT3 treatment and found significant reduction in the expression when compared to non-targeted siRNA. Downstream analysis determined GBM cellular migration was reduced by 40% when treated with GE11-599-siSTAT3.
In conclusion, the GE11-599 tandem peptide forms complexes with siRNA and protects siRNA from degradation. The tandem peptide was cytocompatible in U118 cells and successfully promoted endosomal escape of siRNA. Relative mRNA expression of STAT3 was significantly reduced following treatment with the tandem peptide previously complexed with siSTAT3. To demonstrate the effects of silencing the oncogene STAT3, a scratch on a confluent monolayer showed the groups treated with 60:1 and 80:1 N:P ratios experienced decreased cellular migration. This result confirms the anticipated outcome of silencing STAT3 leading to inhibition metastasis of GBM. Overall, this study shows the feasibility of multifunctional peptides as an RNA delivery agent for use within cancer treatment. Future studies include investigating the capability of the GE11-599 peptide within a mouse model to determine its efficacy in vivo.

Acknowledgements (Optional):  This research was supported in part by the National Science Foundation (NSF) Faculty Early Career Development Program under NSF Award #204669 and NSF EPSCoR Program under NSF Award # OIA-1655740, National Institute of Health (NIH) COBRE Initiative under NIH Award # P30GM131959, MADE in SC, SCBioCRAFT, and Clemson Creative Inquiry.