Engineering Optimized EV-Mimetic Carriers for Efficient Tumor-Targeted Delivery of Functional RNA Nanoparticles

The therapeutic potential of RNAi is limited by the lack of safe, scalable, and efficient delivery systems. Although extracellular vesicles (EVs) are promising carriers for RNA therapeutics, poor scalability and low cargo-loading efficiency constrain their clinical translation. Here, we introduce fully synthetic EV-mimetic (EVM) carriers for the delivery of RNAi-inducing RNA nanoparticles to tumor cells. We systematically evaluated four different EVM cargo-loading strategies using representative RNA nanoparticles and appropriate controls. For each strategy, EVM size, concentration, and cargo encapsulation efficiency were assessed. Cellular uptake and RNA delivery were examined in melanoma 3D spheroids and compared with natural tumor-derived EVs, with EVMs demonstrating superior delivery efficiency. Functional RNAi activation was evaluated in tumor monolayers and 3D spheroids, where EVMs exhibited effective gene silencing with lower toxicity than conventional lipid-based carriers. Overall, our findings establish EVMs as safe and scalable biomimetic platforms for therapeutic nucleic acid delivery and identify optimal loading strategies and RNA nanoparticle designs for achieving maximal gene-silencing.