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Drug Delivery

Drug Delivery - Poster Session C

Poster J2 - Investigating EGFR-targetability of multi-functional peptide to deliver siRNA for treatment of glioblastoma

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

Introduction:  Recent cancer treatment methods have looked to RNA interference (RNAi) as a genetic therapy in which short-interfering RNA (siRNA) degrades specific mRNA to induce gene silencing of an oncogene. However, siRNA has some barriers to delivery due to its size and dense negative charge, requiring a carrier for successful delivery across the cell membrane. The ideal carrier for siRNA protects it from enzymatic degradation, allows for cellular internalization, and promotes endosomal escape of siRNA into the cytosol, where it can be bioactive. Peptides are a promising delivery vehicle for siRNA because of their structural and functional diversity. This study focuses on a targeting peptide and a novel multi-functional peptide comprised of a targeting region and a stimuli-responsive region. The targeting peptide targets the epidermal growth factor receptor (EGFR), which is overexpressed in many cancers, including glioblastoma. This study aims to analyze the internalization and targetability of the targeting sequence within the multi-functional peptide and evaluate its ability to induce gene silencing.

Materials and

Methods:  The peptides used in this study were custom-synthesized custom, purified, and analyzed by Lifetein LLC. Human glioblastoma cell lines, U118MG and U87MG, and human embryonic kidney cell line, HEK293-GFP, were used for these experiments. Fluorescent microscopy analysis and flow cytometry were conducted to evaluate the internalization of siRNA into U118 and U87 cells. A co-culture assay using U87 and HEK293-GFP cells was performed to evaluate the ability of the targeting and multi-functional peptide to target EGFR-expressing cells. A basal expression western blot was completed with these cell lines to confirm EGFR expression. To further investigate EGFR-targetability, an inhibition assay was completed using an anti-EGFR monoclonal antibody to block EGFR and verify the route of internalization of the peptides. RT-qPCR was conducted to analyze the ability of the peptide-siRNA complexes to silence an oncogene.

Results, Conclusions, and Discussions: Uptake images and flow cytometry in U118 and U87 cells indicate successful internalization of siRNA into the cells using each peptide.
To investigate the internalization mechanism, a co-culture assay indicated higher internalization of siRNA into U87 cells than HEK293-GFP cells. A western blot showing more EGFR expression in U87 cells than HEK293-GFP cells, confirms the peptides deliver more siRNA to higher EGFR-expressing cells. To further analyze the ability to target EGFR, an inhibition study was conducted using an anti-EGFR monoclonal antibody to block EGFR, and then peptide-siRNA complexes were delivered to the cells and imaged to determine if there was any internalization while EGFR was blocked. This study confirmed the route of internalization of the multi-functional peptide is the EGFR pathway. Furthermore, RT-qPCR data analyzes the bioactivity of the delivered siRNA by showing efficient knockdown of an oncogene on the mRNA level.
In conclusion, the multi-functional peptide successfully delivers siRNA to U118 and U87 cells. The targeting region of the multi-functional peptide effectively targets EGFR, and the peptide-siRNA system is internalized into the cells via this pathway. The multi-functional peptide can silence oncogene expression, making it a promising cancer therapeutic delivery system. Future studies include in vivo targeting capability, nuclear translocation assays, and downstream analysis of silencing specific oncogenes.

Acknowledgements (Optional):  Funding provided by the National Science Foundation EPSCoR Award #OIA-1655740 and NSF Career Award #2046694.