Programmable multivalent siRNA nanostructure rewires apoptosis by co-silencing MCL-1 and BCL-XL in glioblastoma

Background: Glioblastoma is one of the most aggressive and treatment-resistant brain cancers. Many standard treatments fail because glioblastoma cells rely on powerful survival proteins, especially MCL-1 and BCL-XL, to block cell death, which stabilizes mitochondrial integrity, suppresses caspase activation, and maintains the survival of glioblastoma. Pharmacologic inhibitors of these proteins face dose-limiting toxicities and incomplete target suppression. To overcome these barriers, we engineered a multivalent siRNA nanostructure that enhances functional activation of RNAi modules within tumor cells. Methods: A branched multivalent siRNA nanostructure was first engineered using GFP-targeting siRNAs to validate multivalent RNAi performance in U251-GFP glioblastoma cells. This model enabled assessment of cooperative gene silencing, spatial organization-dependent activity, and prolonged knockdown efficiency. Upon confirming the effectiveness of multivalent RNAi silencing, the nanostructure was redesigned to present siRNAs targeting MCL-1 and BCL-XL, thereby overcoming anti-apoptotic signaling and engaging the intrinsic apoptosis pathways. Functional testing, including qPCR, confocal microscopy, western blotting, mitochondria-dependent apoptosis assays, and long-term knockdown stability, was conducted to evaluate dual-gene silencing and apoptotic activation in U251 glioblastoma cells. Results: The GFP-targeting multivalent siRNA nanostructure exhibited strong cooperative silencing in U251-GFP cells, achieving a sustained reduction in GFP fluorescence of over 90% for up to 7 days, demonstrating enhanced potency, improved intracellular stability, and superior durability compared to a single siRNA. The therapeutic multivalent siRNA nanostructure targets MCL-1 and BCL-XL, achieving robust dual-gene suppression at both mRNA and protein levels. Co-silencing of these anti-apoptotic nodes triggered mitochondrial activation of caspase-9/3 signaling, confirming reactivation of the apoptotic pathway. Cells exhibited a pronounced loss of viability and reduced colony-forming capacity. Conclusions: This study established a programmable multivalent siRNA nanostructure capable of both validating multivalent RNAi using GFP and achieving therapeutic co-targeting of MCL-1 and BCL-XL to overcome apoptotic resistance in glioblastoma. The observed strong apoptotic activation supports the potential of this modular RNAi nanotechnology for treating refractory and malignancies. This approach offers a clinically translatable, programmable strategy for next-generation multi-target RNA interference therapies.