Abstract
INTRODUCTION: Cell membrane-derived nanovesicles, particularly those originating from red blood cells (RNVs), have garnered considerable attention as innovative drug delivery vehicles in oncology, owing to their exceptional biocompatibility, immune evasion, and prolonged systemic circulation. Nevertheless, their inherently poor tumor-targeting efficiency and nonspecific biodistribution present major obstacles to their therapeutic translation. OBJECTIVES: This study sought to functionalize RNVs with a diverse array of tumor-targeting ligands - cRGD, transferrin (TRF), folic acid (FA), GE11, and RVG29 - and to systematically compare their tumor-homing efficiency, biodistribution, and biosafety in a breast cancer model. RESULTS: Functionalized RNVs exhibited markedly enhanced tumor affinity relative to unmodified vesicles in both in vitro and in vivo settings. Among the engineered formulations, RNV@cRGD achieved the most pronounced intratumoral accumulation and cellular uptake, followed sequentially by RNV@GE11, RNV@TRF, RNV@FA, and RNV@RVG29. Fluorescence imaging corroborated the superior tumor selectivity of engineered constructs, all of which also demonstrated robust stability and negligible off-target toxicity in murine models. CONCLUSION: This work presents systematic comparative evaluation of ligand-engineered RNVs, underscoring cRGD as the most potent targeting moiety for breast cancer. These findings illuminate critical design principles for the rational development of tumor-directed RNV-based drug delivery systems and strengthen the translational promise of biomimetic nanocarriers for clinical oncology.