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.

The authors want to make the following corrections to this paper^[1]. In the section “Intracellular trafficking of EVs”, the citations after the sentence “Using another cell-free assay, Morandi et al. provided interesting insights into the process of endosomal fusion in EV cargo release”and “Murphy et al. tackled this question by comparing an FDA-approved cutting-edge lipid nanoparticle (LNP) formulation with EVs in terms of uptake and cargo (specifically gRNA) delivery” were missed. Correctly modify as follows: Using another cell-free assay, Morandi et al. provided interesting insights into the process of endosomal fusion in EV cargo release^[22,68,87]. Murphy et al. tackled this question by comparing an FDA-approved cutting-edge lipid nanoparticle (LNP) formulation with EVs in terms of uptake and cargo (specifically gRNA) delivery^[135]. Corresponding subsequent citation numbers also need to be adjusted: reference 140 is changed to reference 135, reference 135 is changed to reference 136, reference 136 is changed to reference 137, reference 137 is changed to reference 138, reference 138 is changed to reference 139, and reference 139 is changed to reference 140. We apologize for any inconvenience caused and state that the scientific conclusions are unaffected. The original article has been updated.