Engineered Red Blood Cell-Derived Nanovesicles Functionalized with cRGD Peptide and Loaded with Acevaltrate for Targeted Ferroptosis Therapy of Triple-Negative Breast Cancer

Abstract

Triple-negative breast cancer (TNBC) remains a highly aggressive breast cancer subtype with limited molecularly targeted treatment options, while acevaltrate (ACE), a natural dual-target ferroptosis inducer, is constrained by poor aqueous solubility and insufficient tumor accumulation. Here, we engineered cyclic Arg-Gly-Asp (cRGD)-functionalized red blood cell-derived nanovesicles (RNVs) physically encapsulating ACE (cRGD-RNV@ACE) for integrin αvβ3-targeted ferroptosis therapy. The nanovesicles preserved cup-shaped morphology, narrow size distribution, low polydispersity, and favorable blood compatibility. High-performance liquid chromatography confirmed ACE loading in RNV@ACE and cRGD-RNV@ACE, and Fourier transform infrared (FTIR) spectroscopy supported the presence of ACE-associated characteristic bands in RNV@ACE without indicating covalent ACE coupling. cRGD modification enhanced TNBC-cell uptake, and αvβ3 expression analysis together with in vitro competitive uptake assays supported receptor-associated, energy-dependent internalization. cRGD-RNV@ACE showed stronger cytotoxicity than free ACE in MDA-MB-231, MDA-MB-468, and 4T1 cells at equivalent ACE concentrations, accompanied by increased Fe2+ accumulation, lipid peroxidation, GPX4 downregulation, and ferroptosis-inhibitor rescue. In xenograft and immunocompetent 4T1 models, cRGD-RNV@ACE improved tumor accumulation, suppressed tumor growth, prolonged survival, and remodeled the immunosuppressive tumor microenvironment while causing minimal systemic toxicity. These results support cRGD-RNV@ACE as a biomimetic nanotherapeutic platform for targeted TNBC ferroptosis therapy.