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A new study reveals that vesicles released from cell-surface protrusions can deliver active proteins and gene-editing tools far more efficiently than conventional extracellular vesicles, offering new hope for safer medical therapies
Extracellular vesicles (EVs) are tiny membrane-bound particles released by cells to transport proteins and other molecules to neighboring cells. Because of this natural delivery ability, EVs have attracted growing interest as potential vehicles for therapeutic protein and genome-editing enzyme delivery. However, EVs can originate either from intracellular endosomal compartments or directly from specialized protrusions on the cell surface, and until now, it has remained unclear which EV type is more effective at delivering functional protein cargo.
To address this question, researchers in Japan conducted a detailed comparison of these two major EV biogenesis pathways. In a study published in Nature, the team discovered that EVs generated from cell-surface protrusions in an I-BAR protein (MIM)–dependent manner deliver active proteins and genome-editing enzymes far more efficiently than conventional endosome-derived, CD63-associated EVs.
The study was led by Professor Shiro Suetsugu of the Nara Institute of Science and Technology (NAIST). The research team included Associate Professor Tamako Nishimura and then-graduate student Toshifumi Fujioka (NAIST), along with collaborators Professor Takanari Inoue (Johns Hopkins University, USA), Professor Kenichi G. N. Suzuki (Gifu University and National Cancer Center Research Institute, Japan), and Professor Osamu Nureki (The University of Tokyo, Japan).
“In this study, we were able to clearly demonstrate that vesicles generated from cell-surface protrusions function as a highly efficient and natural protein delivery system,” says Suetsugu. “By directly comparing them with endosome-derived vesicles, we gained new insight into how cells achieve efficient cytosolic protein transfer without relying on viral mechanisms and how it can be used in the future.”
To compare the two systems, the researchers cultured human cells and isolated both protrusion-derived EVs and endosome-derived EVs. These vesicles were loaded with defined protein cargo and added to recipient cells. Using advanced live-cell and super-resolution imaging, the team tracked EV uptake via endocytosis, their trafficking through endosomal compartments, and the subsequent release of cargo into the cytoplasm.
The internalization of EVs and transport of Rac1 in endosomes
A Schematic illustration of EV endocytosis, endosomal transport, and cargo release. B 3D-PALM/STORM live cell imaging of the CIP4-mEos4b (magenta)-expressing CIP4 KO PANC-1 cells with l-EV fraction having SF650T-Halo-MIM I-BAR (green). Images were recorded every 10 s. Scale bar, 500 nm. C Colocalization of CIP4-mEos4b with SF650T-Halo-MIM I-BAR in (B). The percentages of voxels co-localized in each frame were calculated (“RAW”). Co-localization of the CIP4 images with the 180°-rotated SF650T-Halo-MIM images is shown as “Randomized”. The 13 super-resolution images from independently seeded 5 cells, which resulted in 4, 1, 4, 2, and 2 images, respectively, were analyzed. D Colocalization of EVs from the l-EV fractions having both Halo-Rac1 and mCherry-MIM I-BAR with EEA1 (Top) or Lamp1 (Bottom) in WT cells after 3 h of incubation, in which 106 cells were treated with 1.6 × 109 EV/ml in 1 ml. Arrowheads in the enlarged images show their colocalization. Scale bars, 10 μm. E Quantification of the colocalization of Halo-Rac1 with EEA1 (Top) or Lamp1 (Bottom) in WT and CIP4 KO cells using Mander’s coefficients after EV addition. The EV fraction and the number of cells and EVs were the same as (D). The red lines indicate the means of 54 cells from 3 independent experiments. F PALM imaging of the l-EVs having SF650B-Halo-Rac1 in the late endosome, having Lamp1-mEos4b (Left). Enlargements of the square regions in the left images are on the Right. Arrowheads indicate possible release of SF650B-Halo-Rac1 from the endosome. Scale bars, 10 μm (Left) and 100 nm (Right). G Quantification of the Halo-Rac1 release from the endosome in (F). The percentage of EVs with crossover of Halo-Rac1 and Lamp1 in a cell is shown (17 cells from 3 independent experiments). All EVs with Lamp1-positive endosomes were counted. The percentage of Halo-Rac1-releasing EVs was calculated as the percentage of “Halo-Rac1-releasing EVs” among the Halo-Rac1 EVs in the Lamp1-positive endosomes (endosome-colocalizing EVs). The means ± SD are shown. Statistical significance was determined by a two-tailed unpaired Mann–Whitney U test (C, E) or a two-tailed t-test with Welch’s correction (G).
One major focus of the study was Rac1, a protein that regulates cell migration. The researchers found that Rac1 delivered by protrusion-derived EVs efficiently escaped from late endosomes into the cytoplasm, where it remained active and stimulated cell movement.
The team also examined whether these EVs could deliver Cas12f, a compact genome-editing enzyme. Protrusion-derived EVs transported Cas12f with dramatically higher functional efficiency—on a per-protein basis—than conventional endosome-derived EVs. Importantly, this high level of genome-editing activity was achieved without the use of viral vectors or viral fusogenic proteins, which are often associated with safety concerns in gene-therapy approaches.
“Our findings show that cells might already possess a remarkably effective, virus-free delivery mechanism,” Suetsugu concludes. “By understanding and harnessing this natural system, we may be able to develop safer and more precise strategies for genome editing, regenerative medicine, and protein-based therapeutics.”
By elucidating how MIM-dependent protrusion-derived EVs protect, transport, and release functional proteins into recipient cells, this study provides a foundation for next-generation delivery technologies that leverage the cell’s own molecular machinery rather than engineered viral components.
Source – Nara Institute of Science and Technology
