ABSTRACT: Rationale：Glioblastoma (GBM) is a highly aggressive brain tumor, often associated with the activation of EGFR and NF-κB signaling pathways in radioresistant GBM cells. Concurrently targeting these pathways presents a promising strategy to overcome radioresistance and enhance therapeutic efficacy. Methods：We established a radioresistant glioblastoma stem cell (GSCs) model by exposing U87-derived GSCs to 5 Gy irradiation for 13 cycles. After differentiation and colony formation selection, we obtained a radioresistant cell line (Diff cells). These model cell lines mimic the progression of recurrence in GBM. Transcriptomic analysis of Diff cells, GSCs, and U87 cells, combined with public datasets from The Cancer Genome Atlas (TCGA), was conducted to identify key signaling pathways involved in radioresistance. Based on these findings, we designed a novel dual-functionalized nanoliposome (iRGD-OB-LP) for the co-delivery of Osimertinib and Bortezomib to simultaneously inhibit the EGFR and NF-κB pathways. The physicochemical properties, cellular uptake, and drug release profiles of iRGD-OB-LP were characterized, and its targeting capability, antitumor activity, and radiosensitization potential were evaluated both in vitro and in vivo. Results: We successfully established GSCs from U87 cells and induced radioresistance in these GSCs. The Diff cells derived from GSCs retained radioresistant characteristics and exhibited significant resistance to radiotherapy-induced cell death. Transcriptomic analysis of Diff cells, GSCs, and U87 cells, along with TCGA data, identified EGFR and NF-κB (RelA/p65) as key pathways involved in radioresistance. iRGD-OB-LP demonstrated efficient tumor-specific targeting, cellular uptake, and sustained drug release. In vitro, iRGD-OB-LP effectively inhibited EGFR and NF-κB pathway activation, prolonged DNA damage induced by radiotherapy, reduced tumor cell stemness, and enhanced radiotherapy-induced apoptosis. In vivo, studies using an orthotopic GBM mouse model revealed that iRGD-OB-LP successfully crossed the blood-tumor barrier, accumulated at tumor sites, and synergistically enhanced the efficacy of radiotherapy, as evidenced by suppressed tumor growth and prolonged survival. Conclusion: In this study, we established Diff cells as a radioresistant model derived from GSCs and identified EGFR and NF-κB (RelA/p65) as critical pathways in sustaining the radioresistant phenotype through RNA sequencing and TCGA data analysis. The iRGD-OB-LP nanoplatform offers a promising strategy for targeted delivery of dual inhibitors to simultaneously inhibit EGFR and NF-κB, overcoming GBM radioresistance. This innovative approach highlights a potential strategy to improve radiotherapy outcomes in GBM treatment.
