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

Ning Ding; Armond Daci; Vanesa Krasniqi; Rachel Butler; Alan Goddard; Qing Guo; Yunyue Zhang; Jizhou Zhong; K L Andrew Chan; Maya Thanou; Driton Vllasaliu

doi:10.1016/j.ijpx.2025.100428

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

Ning Ding
, Armond Daci
, Vanesa Krasniqi
, Rachel Butler
, Alan Goddard
, Qing Guo
, Yunyue Zhang
, Jizhou Zhong
, K L Andrew Chan
, Maya Thanou
, Driton Vllasaliu

Institute of Pharmaceutical Science, School of Cancer and Pharmaceutical Science, King's College London, London SE1 9NH, United Kingdom.
Department of Pharmacy, Faculty of Medicine, University of Prishtina, Prishtina 10000, Kosovo.

Research output: Contribution to journal › Article › peer-review

2 Citations (SciVal)

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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.

Original language	English
Article number	100428
Number of pages	13
Journal	International Journal of Pharmaceutics: X
Volume	10
Early online date	26 Oct 2025
DOIs	https://doi.org/10.1016/j.ijpx.2025.100428
Publication status	Published - 1 Dec 2025

Bibliographical note

Data Access Statement

Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijpx.2025.100428. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Funding

This work was funded by the King’s-China Scholarship Council PhD Scholarship Programme, the European Union (EuropeAid, grant no. EUROPEAID/173691/DD/ACT/XK) and BBSRC Engineering Biology Mission Award (BB/Y008065/1). The Aston Institute for Membrane Excellence (AIME) is funded by UKRI’s Research England as part of their Expanding Excellence in England (E3) fund.

Funders	Funder number
European Commission
China Scholarship Council
UK Research and Innovation
EuropeAid	EUROPEAID/173691/DD/ACT/XK
Biotechnology and Biological Sciences Research Council	BB/Y008065/1

Keywords

Sirna Delivery
Rna Delivery
Extracellular Vesicles (Evs)
Ev-Lnp Hybrids
Oral Rna Therapy

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10.1016/j.ijpx.2025.100428Licence: CC BY 4.0

Engineered extracellular vesicles demonstrate altered endocytosis and biodistribution and have superior oral siRNA delivery efficiency compared to lipid nanoparticles
Copyright © 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
Final published version, 5.3 MBLicence: CC BY 4.0

Cite this

Ding, N., Daci, A., Krasniqi, V., Butler, R., Goddard, A., Guo, Q., Zhang, Y., Zhong, J., Chan, K. L. A., Thanou, M., & Vllasaliu, D. (2025). Engineered extracellular vesicles demonstrate altered endocytosis and biodistribution and have superior oral siRNA delivery efficiency compared to lipid nanoparticles. International Journal of Pharmaceutics: X, 10, Article 100428. https://doi.org/10.1016/j.ijpx.2025.100428

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title = "Engineered extracellular vesicles demonstrate altered endocytosis and biodistribution and have superior oral siRNA delivery efficiency compared to lipid nanoparticles",

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.",

keywords = "Sirna Delivery, Rna Delivery, Extracellular Vesicles (Evs), Ev-Lnp Hybrids, Oral Rna Therapy",

author = "Ning Ding and Armond Daci and Vanesa Krasniqi and Rachel Butler and Alan Goddard and Qing Guo and Yunyue Zhang and Jizhou Zhong and Chan, \{K L Andrew\} and Maya Thanou and Driton Vllasaliu",

note = "Copyright {\textcopyright} 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).",

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Ding, N, Daci, A, Krasniqi, V, Butler, R, Goddard, A, Guo, Q, Zhang, Y, Zhong, J, Chan, KLA, Thanou, M & Vllasaliu, D 2025, 'Engineered extracellular vesicles demonstrate altered endocytosis and biodistribution and have superior oral siRNA delivery efficiency compared to lipid nanoparticles', International Journal of Pharmaceutics: X, vol. 10, 100428. https://doi.org/10.1016/j.ijpx.2025.100428

TY - JOUR

T1 - Engineered extracellular vesicles demonstrate altered endocytosis and biodistribution and have superior oral siRNA delivery efficiency compared to lipid nanoparticles

AU - Ding, Ning

AU - Daci, Armond

AU - Krasniqi, Vanesa

AU - Butler, Rachel

AU - Goddard, Alan

AU - Guo, Qing

AU - Zhang, Yunyue

AU - Zhong, Jizhou

AU - Chan, K L Andrew

AU - Thanou, Maya

AU - Vllasaliu, Driton

PY - 2025/12/1

Y1 - 2025/12/1

N2 - 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.

AB - 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.

KW - Sirna Delivery

KW - Rna Delivery

KW - Extracellular Vesicles (Evs)

KW - Ev-Lnp Hybrids

KW - Oral Rna Therapy

UR - https://www.sciencedirect.com/science/article/pii/S2590156725001136

UR - https://www.scopus.com/pages/publications/105020968218

U2 - 10.1016/j.ijpx.2025.100428

DO - 10.1016/j.ijpx.2025.100428

M3 - Article

SN - 2590-1567

VL - 10

JO - International Journal of Pharmaceutics: X

JF - International Journal of Pharmaceutics: X

M1 - 100428

ER -

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

Abstract

Bibliographical note

Data Access Statement

Funding

Keywords

Access to Document

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