Split-intein-mediated engineering of VSV-G-free extracellular vesicles for enhanced cargo loading and delivery of functional proteins

Extracellular Vesicles (EVs) are a heterogeneous group of membranous nanoparticles. Due to their unique properties, they are considered promising nanocarriers for therapeutic applications. Cargo loading and functional delivery to the target cell are challenging areas and are receiving increasing attention in EV research. Here, we introduce a novel one-step and VSV-G-free loading system based on pH-sensitive split inteins to improve protein loading into EVs. To this end, we fused the EV membrane protein CD9 to the NpuN component and the cargo of interest to the NpuC component. We reasoned that the interaction between NpuN and NpuC facilitates loading of the fused cargo into the EVs. We expected that the pH-sensitive sequence between NpuC and the cargo would cleave at acidic pH within the intraluminal vesicles of producing cells or in the endosomes of recipient cells. Our results showed that the NpuN/NpuC system, compared with passive loading, improved the loading of DsRed2 and tdTomato into the EVs by 1.3-fold. We employed this system to assess the functional delivery of proteins into recipient cells. To this end, piggyBac transposase was fused to NpuC, and EVs were added to reporter cells, where the excisionase activity of piggyBac was consistent with the excision of 5′ITR-EGFP-Polyadenylation signal-3′ITR and expression of mCherry. Isolated EVs were characterized by TEM, DLS, and dot blot detection of the engineered mEmerald-CD9 membrane protein and DsRed2 cargo in the vesicle fraction. No endogenous EV markers or negative-contamination markers were assessed; consequently, EV identity and sample purity were not established by protein marker profiling.