Background/Objectives: RNA interference (RNAi) represents a promising therapeutic approach for silencing oncogenes involved in cancer progression by utilizing small interfering RNA (siRNA). However, siRNA requires an efficient delivery system to overcome cellular uptake and endosomal escape barriers. This study aimed to evaluate a multifunctional tandem peptide, GE11-599, designed to enhance the targeted delivery of siRNA and maintain its bioactivity in glioblastoma (GBM) cells. Methods: The GE11-599 peptide, consisting of an EGFR-targeting GE11 motif and a 599 fusogenic domain, was complexed with siRNA via electrostatic interactions to form nanoparticles. We assessed nanoparticle physicochemical properties, protection of siRNA from serum and RNase degradation, and cellular uptake in two GBM cell lines (U118MG and U87MG). Mechanistic studies evaluated receptor-mediated endocytosis and the subsequent escape from endosomes. Functional assays quantified STAT3 gene silencing and downstream effects on cell migration following treatment with GE11-599-siSTAT3 complexes. Results: GE11-599 formed positively charged, monodisperse nanoparticles capable of protecting siRNA from degradation. The tandem peptide significantly enhanced cellular internalization through EGFR-mediated endocytosis and facilitated endosomal escape of siRNA. Treatment with GE11-599-siSTAT3 resulted in robust gene silencing, achieving up to an 80% reduction in STAT3 mRNA expression. Downstream functional assessment showed a 40% decrease in migration in GBM cells treated with GE11-599-siSTAT3 complexes. Conclusions: The GE11-599 tandem peptide effectively enhances cell-specific internalization and endosomal escape of siRNA in GBM cells, resulting in increased siRNA bioactivity and functional gene silencing. These findings support GE11-599 as a promising siRNA delivery platform for targeting EGFR-expressing cancers.
