Title : NPY-Functionalized erythrocyte vesicles for targeted breast cancer therapy
Abstract:
Current therapeutic strategies for breast cancer often lack tumor specificity, leading to off-target effects and systemic toxicity. The Y1 receptor (NPYR1), a subtype of the Neuropeptide Y receptor family, is highly expressed in 100% of NPYR-positive breast tumors but only in 58% of healthy breast tissue. This makes it a promising target for selective drug delivery. Erythrocyte membrane vesicles (EMVs) are ideal for use as biomimetic nanocarriers due to their native membrane proteins, capacity for immune evasion, and prolonged circulation time. We report the development of NPY-functionalized extracellular membrane vesicles (NPY-EMVs) designed to enhance receptor-targeted delivery in breast cancer treatment. EMVs were prepared from fresh human erythrocytes through hypotonic lysis, followed by sonication and extrusion. A comparative analysis of fresh and frozen erythrocytes was conducted to assess protein preservation. We evaluated samples from whole cells, cell ghosts, EMVs, and EMVs purified by ultracentrifugation. Proteomic profiling was performed using SDS-PAGE, in-gel digestion, and nano LC- MS/MS. Western blot analysis confirmed the presence of critical membrane proteins, including CD47, CD35, and Band 3. NPY analogs, created through molecular modeling and available with or without FITC at various positions, were conjugated using maleimide-thiol chemistry. Transmission electron microscopy (TEM) confirmed the morphology of the vesicles. The EMVs retained key membrane proteins, indicating successful preservation during preparation. In contrast, the frozen erythrocytes exhibited significant protein loss, reducing the integrity of the vesicles. We are optimizing the purification process to enhance conjugation efficiency and minimize peptide loss. NPY-functionalized EMVs represent a stable and modular platform for targeted therapy against the Y1 receptor in breast cancer. The preservation of protein profiles, effective peptide conjugation, and changes in vesicle properties support their application for receptor-mediated drug delivery. This approach enables future therapeutic loading and precise targeting, laying the groundwork for biomimetic nanocarriers in precision oncology.
