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Ribovski et al. Extracell Vesicles Circ Nucleic Acids 2023;4:283-305 Extracellular Vesicles and
DOI: 10.20517/evcna.2023.26
Circulating Nucleic Acids

Review Open Access

Breaking free: endocytosis and endosomal escape
of extracellular vesicles

1,#
1
2,#
Laís Ribovski , Bhagyashree Joshi , Jie Gao , Inge Zuhorn 1
1
Department of Biomedical Engineering, University Medical Center Groningen, University of Groningen, Groningen 9713 AV, the
Netherlands.
2
Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft 2629 HZ, the Netherlands.
#
Authors contributed equally.
Correspondence to: Dr. Inge S. Zuhorn,Department of Biomedical Engineering, University Medical Center Groningen, University
of Groningen, A. Deusinglaan 1, Groningen 9713 AV, the Netherlands, E-mail: i.zuhorn@umcg.nl
How to cite this article: Ribovski L, Joshi B, Gao J, Zuhorn I. Breaking free: endocytosis and endosomal escape of extracellular
vesicles. Extracell Vesicles Circ Nucleic Acids 2023;4:283-305. https://dx.doi.org/10.20517/evcna.2023.26

Received: 26 Apr 2023 First Decision: 18 May 2023 Revised: 17 Jun 2023 Accepted: 25 Jun 2023 Published: 30 Jun 2023

Academic Editor: Yoke Peng Loh Copy Editor: Yanbing Bai Production Editor: Yanbing Bai

Abstract
Extracellular vesicles (EVs) are natural micro-/nanoparticles that play an important role in intercellular
communication. They are secreted by producer/donor cells and subsequent uptake by recipient/acceptor cells
may result in phenotypic changes in these cells due to the delivery of cargo molecules, including lipids, RNA, and
proteins. The process of endocytosis is widely described as the main mechanism responsible for cellular uptake of
EVs, with endosomal escape of cargo molecules being a necessity for the functional delivery of EV cargo.
Equivalent to synthetic micro-/nanoparticles, the properties of EVs, such as size and composition, together with
environmental factors such as temperature, pH, and extracellular fluid composition, codetermine the interactions
of EVs with cells, from binding to uptake, intracellular trafficking, and cargo release. Innovative assays for detection
and quantification of the different steps in the EV formation and EV-mediated cargo delivery process have provided
valuable insight into the biogenesis and cellular processing of EVs and their cargo, revealing the occurrence of EV
recycling and degradation, next to functional cargo delivery, with the back fusion of the EV with the endosomal
membrane standing out as a common cargo release pathway. In view of the significant potential for developing EVs
as drug delivery systems, this review discusses the interaction of EVs with biological membranes en route to cargo
delivery, highlighting the reported techniques for studying EV internalization and intracellular trafficking, EV-
membrane fusion, endosomal permeabilization, and cargo delivery, including functional delivery of RNA cargo.
Keywords: Extracellular vesicles, endocytosis, intracellular trafficking, functional delivery, endosomal escape

© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0
International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, sharing,
adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, as
long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and
indicate if changes were made.

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