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

Rationale: 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. Methods: 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. Results: 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. Conclusions: 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.