L-lysine α-oxidase Encapsulation in Red Blood Cells - Derived Extracellular Vesicles as Biocompatible Nanoplatform for Cancer Therapy

Amino acid-degrading enzyme-based cancer therapy faces significant challenges such as rapid clearance, immunogenicity, and systemic toxicity. This study introduces red blood cell-derived extracellular vesicles (RBCEVs) as a transformative platform for the encapsulation, stabilization, and controlled delivery of L-lysine α-oxidase (LO; EC 1.4.3.14), an enzyme that depletes tumor-essential L-lysine (L-lys) while generating cytotoxic hydrogen peroxide. RBCEVs effectively encapsulate LO with high efficiency, retaining 91% of enzymatic activity post-release in vitro. They achieve sustained plasma L-lys depletion for 24 hours followed by full metabolic recovery within seven days, striking a balance between efficacy and safety. Fluorescence-conjugated LO confirms cargo loading and vesicle integrity, while RBCEVs' monodisperse size (∼200 nm), anionic charge (-16.5 mV), and long-term stability at 4°C underscore their clinical viability. Unlike synthetic carriers, RBCEVs offer a modular platform for diverse therapeutic enzymes, circumventing immunogenicity risks and overcoming barriers in enzyme delivery, paving the way for clinical use.