Single-Particle Deconvolution of Electroporation Artifacts Guides the Rational Engineering of CD47-EV–Liposome Hybrids for siRNA Delivery

The clinical translation of small interfering RNA (siRNA) therapeutics is hampered by the lack of efficient delivery systems. Extracellular vesicles (EVs) hold great promise as natural carriers, yet conventional loading methods, most notably electroporation, suffer from low encapsulation efficiency, structural damage, and poorly characterized aggregation artifacts. Using single-particle nano-flow cytometry, we deconvoluted these electroporation-induced artifacts and revealed that true loading efficiency is only ∼24%. To overcome these limitations, we engineered CD47-functionalized EV–liposome hybrid vesicles via membrane fusion of CD47-overexpressing EVs with siRNA-loaded liposomes. The hybrid design integrates the high payload capacity of synthetic liposomes with the intrinsic biocompatibility and immune-evasive functionality of CD47-engineered EVs. In a Transwell coculture model simulating the competitive uptake environment of the tumor microenvironment, the hybrid vesicles evaded macrophage clearance and delivered anti-PD-L1 siRNA to tumor cells, resulting in ∼60% PD-L1 gene silencing, three times that of electroporation (∼20%). By providing a rationally designed hybrid nanocarrier with validated immune-evasive and RNAi performance, this work offers a significant advancement for EV-based RNA interference (RNAi) therapeutics.