An Easy Oligonucleotide Platform for Rapid, Post-Isolation EV Modification: Dual-Function Uptake Control and Enhanced Targeting

Extracellular vesicles (EVs) hold immense therapeutic potential, yet their clinical translation is hindered by nonspecific cellular uptake and the complexity and cost of surface engineering. Here, we present the EZON (Easy OligoNucleotide) EV modification platform, a cost-effective, post-isolation modification system that addresses both limitations simultaneously. The EZON platform employs unmodified DNA aptamers that bind directly to tetraspanin proteins on EV surfaces, eliminating the need for cholesterol conjugation, covalent chemistry, or genetic engineering of producer cells. EVs are thus easily and rapidly functionalized in a single incubation step at substantially reduced oligonucleotide cost. The EZON platform enables dual functionality: (1) the oligonucleotide anchor (ONA) sequence inhibits nonspecific cellular uptake, while (2) the display strand enables targeted delivery through ligand-mediated internalization. We demonstrate that our optimized ONA construct efficiently functionalizes EVs from multiple mammalian cell types. Cancer cells selectively internalized EZON-modified EVs functionalized with AS1411, an aptamer that targets nucleolin, whereas off-target internalization was suppressed. The platform's versatility extends to protein display via histidine-tag aptamers and therapeutic cargo delivery, as demonstrated by targeted doxorubicin delivery to head and neck squamous cell carcinoma cells. This rapid, modular approach transforms unmodified EVs into precision delivery vehicles within hours, offering a scalable solution for targeted therapeutics.