Engineered RAP-anchored copper-escorting liposomes for FDX1-targeted cuproptosis in glioblastoma therapy‌.

Glioblastoma multiforme (GBM), the most aggressive primary brain malignancy, presents considerable therapeutic challenges due to intrinsic treatment resistance and dismal clinical outcomes. Capitalizing on emerging insights into cuproptosis-mediated oncotherapy, we have developed a receptor-associated protein (RAP)-modified liposomal nanoplatform (RAP-LPs@ESCu) for the precise delivery of elesclomol-copper complexes (ESCu) and aimed to evaluate its therapeutic potential in triggering tumor-specific cuproptosis. RAP-LPs@ESCu were synthesized via thin-film hydration and characterized by transmission electron microscope (TEM) and dynamic light scatting. Intracellular copper levels were quantified via atomic absorption spectroscopy. RNA sequencing was used to identify cuproptosis-related molecular targets, among which ferredoxin-1 (FDX1) was the primary focus of the study. Western blot, immunohistochemistry, immunofluorescence, flow cytometry and biochemical kits were applied to elucidate the molecular mechanism of cuproptosis triggered by RAP-LPs@ESCu. Orthotopic GBM models were established by stereotactic implantation of luciferase-labeled LN229 cells into the right striatum of BALB/c-nu mice. In vivo imaging system was utilized to monitor tumor progression and blood-brain barrier (BBB) penetration. Copper content in tumor tissues was quantified by biochemical kit, and mitochondrial morphology was examined by TEM. Systemic toxicity of RAP-LPs@ESCu was evaluated through hematological, biochemical, hemolysis assays, and hematoxylin-eosin staining. Neurological and motor functions were assessed using the Loga 5-score test and open-field test. Through systematic evaluation in an orthotopic xenograft mouse model, we found that RAP-LPs@ESCu effectively induced cuproptosis, inhibited GBM progression, and significantly prolonged survival. Mechanistic studies revealed that RAP-mediated targeting resulted in efficient BBB penetration and preferential accumulation of ESCu in tumor cells. Subsequent intracellular Cu²⁺ overload triggered a cascade of molecular events beginning with substantial upregulation of FDX1 expression, followed by accumulation of lipoylated dihydrolipoamide S-acetyltransferase aggregates, and finally depletion of iron-sulfur cluster proteins. These coordinated effects culminated in the selective induction of cuproptosis in GBM cells. This study successfully constructed RAP-LPs@ESCu which selectively eliminated mitochondria-metabolically active GBM cells via an FDX1-dependent cuproptosis pathway, ultimately achieving orthotopic GBM growth suppression.