Background <p>Systemic toxicity and limited tumor selectivity remain major obstacles in HER2-positive breast cancer therapy with conventional chemotherapeutics. Extracellular vesicles (EVs) provide a promising delivery platform due to their intrinsic biocompatibility and intercellular communication. Here, we engineered EVs displaying the HER2-binding peptide P51 to create a peptide-mediated targeted delivery system for doxorubicin (Dox), aiming to enhance tumor specificity and apoptotic efficacy.</p> Methods <p>EVs expressing the P51 peptide were generated by transient transfection of HEK-293 cells, followed by Dox loading to produce P51-EV^Dox. Cellular uptake and antitumor efficacy were evaluated in vitro and in vivo using HER2-overexpressing BT-474 breast cancer cells. RNA sequencing and apoptosis-related protein analyses, including cleaved caspase-3, PARP cleavage, and p53 expression, were performed to elucidate EV-mediated molecular mechanisms.</p> Results <p>P51-EV^Dox exhibited significantly enhanced binding and intracellular accumulation in HER2-positive cells compared with non-targeted EV-Dox. Transcriptomic analysis comparing free Dox and P51-EV^Dox revealed marked upregulation of thioredoxin-interacting protein (TXNIP). Consistently, protein-level analyses demonstrated increased activation of apoptosis-associated markers, including cleaved caspase-3, PARP cleavage, and p53, following P51-mediated EV delivery. These findings suggest activation of a ROS/TXNIP-driven apoptotic cascade.</p> Conclusions <p>This study demonstrates that peptide-functionalized EVs represent a promising tumor-targeted chemotherapeutic platform for HER2-positive breast cancer. By integrating ligand-mediated cellular recognition with EV-based doxorubicin delivery, P51-EV^Dox activates the ROS/TXNIP signaling pathway, leading to enhanced tumor suppression and apoptosis.</p>