Worm-Like Nanostructure and Protrusion-Derived EVs Advance Gene and Protein Delivery
A worm-like nanostructure uses the ClC3 ion exchanger for endosomal escape; protrusion-derived extracellular vesicles deliver proteins and genome-editing enzymes more efficiently than endosome-derived EVs. Both avoid viral vectors.
Researchers have developed a worm-like nucleic acid nanostructure for efficient gene delivery and endosome escape, and separately discovered that extracellular vesicles generated from cell-surface protrusions deliver active proteins and genome-editing enzymes far more efficiently than conventional endosome-derived vesicles. Both approaches address the challenge of endosomal escape, a key bottleneck in non-viral gene and protein delivery.
Nanoparticles are established gene carriers with low immunogenicity relative to viral vectors, but their endosomal escape remains inefficient. Since 2019, there have been only 14 of >38,000 research articles reporting a correlation coefficient <0.2 between the NP-gene complex and endolysosomes. The two mainstream gene carriers for endosomal escape are cationic NPs and lipid-based NPs. Cationic NPs escape endosomes via the 'proton sponge effect,' but the positive charge often induces cytotoxicity. Lipid-based NPs escape via 'membrane destabilization,' yet lipid screening can be challenging and lipids can induce membrane damage and inflammation.
Nucleic acid nanostructures, including spherical nucleic acid, origami, tile-based nanostructures, and nucleic acid nanogels, offer a promising alternative with biocompatibility, transfection-free cellular uptake, and mitigated cytotoxicity and immunogenicity, but their endosomal escape is inefficient and often requires cationic groups, cell-penetrating peptides, or mechanical stimuli. The new nanostructure adsorbs therapeutic nucleic acids onto the surface of a structural template made of a hybrid gold-polydopamine core-shell nanoworm (Au@PDA NW). The overall nanostructure is anionic, enters cells without transfection agents, and escapes endosomes with a Pearson correlation coefficient <0.2 in four cell types, all without cationic/lipidic functional groups or mechanical stimuli. It naturally activates the chloride voltage-gated channel 3 (ClC3) ion exchanger, which mediates vesicular accumulation of H+ and Cl- and endosomal escape.
The nanostructure enabled in vitro miRNA-enabled macrophage polarization and siRNA-enabled stromal cell differentiation, ex vivo mRNA-enabled cell-based therapy for reducing kidney fibrosis, and in vivo mRNA delivery to hepatocytes for treating liver injury, outperforming Lipofectamine in endosomal escape and efficacy.
In a study published in Nature Communications on December 8, 2025, researchers in Japan compared the two major extracellular vesicle biogenesis pathways. Extracellular vesicles are tiny membrane-bound particles released by cells to transport proteins and other molecules to neighboring cells. They can originate either from intracellular endosomal compartments or directly from specialized protrusions on the cell surface. 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 researchers cultured human cells and isolated both protrusion-derived EVs and endosome-derived EVs, loaded them with defined protein cargo, and added them to recipient cells. Using live-cell and super-resolution imaging, they tracked EV uptake, trafficking through endosomal compartments, and release of cargo into the cytoplasm. They found that Rac1, a protein that regulates cell migration, delivered by protrusion-derived EVs efficiently escaped from late endosomes into the cytoplasm, remained active, and stimulated cell movement. The team also showed that protrusion-derived EVs transported Cas12f, a compact genome-editing enzyme, with dramatically higher functional efficiency on a per-protein basis than conventional endosome-derived EVs, without the use of viral vectors or viral fusogenic proteins.
'Our findings show that cells might already possess a remarkably effective, virus-free delivery mechanism,' the lead researcher said, adding that harnessing this natural system could lead to safer and more precise strategies for genome editing, regenerative medicine, and protein-based therapeutics. The study provides a foundation for next-generation delivery technologies that leverage the cell's own molecular machinery rather than engineered viral components.