# Engineered extracellular vesicles demonstrate altered endocytosis and biodistribution and have superior oral siRNA delivery efficiency compared to lipid nanoparticles

## TL;DR

Researchers developed engineered extracellular vesicles that improve oral RNA delivery efficiency and reduce toxicity compared to traditional methods.

## Contribution

A microfluidic platform was used to create EV-LNP hybrids with superior oral RNA delivery and intestinal targeting.

## Key findings

- EV-LNP hybrids showed 40–60% gene knockdown in Caco-2 cells after intestinal fluid exposure.
- Hybrids accumulated preferentially in the colon of mice compared to native EVs or LNPs.
- EV-LNP hybrids demonstrated lower cytotoxicity and improved intestinal transport.

## Abstract

Oral administration of RNA therapeutics remains a major unsolved challenge due to currently insurmountable biological barriers. Extracellular vesicles (EVs) are natural carriers capable of traversing the intestinal barrier, but inefficient RNA loading into EVs in general severely limits the application of EVs for RNA delivery. Here, we utilize a microfluidic engineering platform to generate milk-derived EV-lipid nanoparticle (EV-LNP) hybrids for oral delivery of RNA. The process produced uniform nanoparticles (133 nm, polydispersity index 0.19) with >45 % dual-positive fusion efficiency, significantly outperforming freeze–thaw hybridization. Compared to conventional LNPs, EV-LNP hybrids exhibited lower cytotoxicity, altered epithelial uptake pathways, and markedly improved intestinal epithelial transport. Importantly, the hybrids retained gene-silencing efficacy following exposure to simulated intestinal fluids, achieving 40–60 % glyceraldehyde 3-phosphate dehydrogenase knockdown in Caco-2 cells, which was superior to LNPs. Oral gavage in mice revealed preferential colonic accumulation of EV-LNP hybrids compared to native EVs or LNPs, indicating strong potential for local RNA therapy in gut diseases such as colitis. Collectively, this study establishes a scalable, bioinspired delivery platform that addresses key translational barriers for oral RNA therapeutics and enables targeted delivery to the colon.

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