(F-220) A Targeted Stimuli-Responsive Peptide System for siRNA Delivery for Glioblastoma Multiforme Treatment

Introduction:: Glioblastoma Multiforme (GBM is a grade VI glioma, a highly aggressive malignant tumor associated with a poor prognosis. Its incidence rate is 3.19 per 100,000, and GBM has a median survival rate of 12-15 months and a five-year survival rate of less than 5%. While typical treatments involve surgical removal, chemotherapy, and radiation, they still result in low survival rates and potentially harm healthy cells. To target oncogenes for cancer treatment, RNAi, a naturally occurring process, utilizes siRNA for sequence-specific mRNA for degradation to inhibit protein translation. However, siRNA delivery faces significant barriers, such as insufficient cellular uptake, non-specific uptake, and lack of endosomal escape, which hinders clinical translation. Peptides show promise as a carrier for siRNA as they can overcome these delivery barriers. In this study, a novel targeted stimuli-responsive peptide, or tandem peptide, is designed for siRNA delivery and evaluated for its ability to mediate the knockdown of a target oncogene. The tandem peptide combines a targeting peptide for specific delivery to GBM cells and a fusogenic peptide for mediating endosomal escape of the bioactive siRNA into the cytoplasm. The target oncogene, STAT3, plays a role in cell proliferation, differentiation, apoptosis, and angiogenesis in GBM. Therefore, the hypothesis is that the tandem peptide will enhance the efficacious delivery of siRNA into glioblastoma cells, resulting in more effective gene silencing and associated downstream effects compared to the fusogenic and targeting peptides alone.


Materials and Methods:: The three peptides used in this study were custom synthesized, purified, and analyzed by GenScript USA Inc. Human glioblastoma cells lines, U118MG and U87MG, obtained from American Type Culture Collection (ATCC) were used for these experiments. Characterization of the peptide/siRNA complexes was conducted via dynamic light scattering (DLS) using a zetasizer. Gel shift assays were performed to determine the ability of the peptides to complex with siRNA and protect the siRNA at physiological conditions. MTS viability assays were performed to evaluate changes in cell viability following peptide transfection. The ability of the peptide/siRNA complexes to be internalized into the cell was assessed through fluorescent imaging and flow cytometry. qPCR and western blot experiments analyze the silencing efficiency of the peptide/siRNA complexes on the mRNA and protein levels, respectively.


Results, Conclusions, and Discussions:: Western Blot results confirmed basal expression of target genes, STAT3 and EGFR, in GBM cell lines. Characterization via DLS of all three peptides indicated the formation of positive-charged, monodisperse nanoparticles with diameters less than 200 nm with siRNA complexation. Furthermore, gel shift assays confirmed tandem peptide complexation and protection of siRNA at 20-80:1 N:P ratios. A summary of characterization data is shown in Figure 1a. MTS viability assays conducted in cell lines for each peptide confirmed no significant cytotoxicity due to the vehicles alone. Fluorescent uptake images and flow cytometry confirmed enhanced uptake of the tandem peptide in the U87 and U118 cell lines compared to the control cells. The tandem peptide was complexed with STAT3 siRNA and transfected into GBM cells to determine gene silencing capability. qPCR analysis shows the tandem peptide’s ability to reduce the STAT3 mRNA, especially at a 60:1 ratio (Figure 1b). These experiments were also used to compare the efficiency of the tandem peptide to the individual sequences, with results showing the tandem peptide having enhanced efficacy.



In conclusion, results have confirmed characteristics of the tandem peptide necessary for the effective delivery of bioactive siRNA, including complete complexation with siRNA, formation of monodisperse particles, and the ability to protect siRNA from degradation. This work investigated the in vitro analysis of the tandem peptide, indicating enhanced cell internalization, no cytotoxicity to the cell lines, and efficient gene silencing of our oncogene mRNA. Future studies include further gene silencing studies using Western Blot and qPCR assays and downstream analysis of silencing the STAT3 oncogene, such as migration and invasion assays.


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


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