BACKGROUND: Extracellular vesicles (EVs) are cell-secreted particles conceived as natural vehicles for intercellular communication. The capacity to entrap heterogeneous molecular cargoes and target specific cell populations through EV functionalization promises advancements in biomedical applications. However, the efficiency of the obtained EVs, the contribution of cell-exposed receptors to EV interactions, and the predictability of functional cargo release with potential sharing of high molecular weight recombinant mRNAs are crucial for advancing heterologous EVs in targeted therapy applications. METHODS: In this work, we selected the popular EV marker CD81 as a transmembrane guide for fusion proteins with a C-terminal GFP reporter encompassing or not Trastuzumab light chains targeting the HER2 receptor. We performed high-content imaging analyses to track EV-cell interactions, including isogenic breast cancer cells with manipulated HER2 expression. We validated the functional cargo delivery of recombinant EVs carrying doxorubicin upon EV-donor cell treatment. Then, we performed an in vivo study using JIMT-1 cells commonly used as HER2-refractory, trastuzumab-resistant model to detect a more than 2000 nt length recombinant mRNA in engrafted tumors. RESULTS: Fusion proteins participated in vesicular trafficking dynamics and accumulated on secreted EVs according to their expression levels in HEK293T cells. Despite the presence of GFP, secreted EV populations retained a HER2 receptor-binding capacity and were used to track EV-cell interactions. In time-frames where the global EV distribution did not change between HER2-positive (SK-BR-3) or -negative (MDA-MB-231) breast cancer cell lines, the HER2 exposure in isogenic cells remarkably affected the tropism of heterologous EVs, demonstrating the specificity of antiHER2 EVs representing about 20% of secreted bulk vesicles. The specific interaction strongly correlated with improved cell-killing activity of doxorubicin-EVs in MDA-MB-231 ectopically expressing HER2 and reduced toxicity in SK-BR-3 with a knocked-out HER2 receptor, overcoming the effects of the free drug. Interestingly, the fusion protein-corresponding transcripts present as full-length mRNAs in recombinant EVs could reach orthotopic breast tumors in JIMT-1-xenografted mice, improving our sensitivity in detecting penetrant cargoes in tissue biopsies. CONCLUSIONS: This study highlights the quantitative aspects underlying the creation of a platform for secreted heterologous EVs and shows the limits of single receptor-ligand interactions behind EV-cell engagement mechanisms, which now become the pivotal step to predict functional tropism and design new generations of EV-based nanovehicles.