Cellular uptake of extracellular vesicles is mediated by clathrin-independent endocytosis and macropinocytosis

TLDR

Extracellular vesicles (EVs) act as an endogenous transport system for biomolecules, offering significant potential for drug delivery and regenerative medicine, making their intercellular transfer biology crucial for therapeutic development. The study aimed to characterize the cellular mechanisms of EV uptake and to identify EV components that facilitate functional cargo transfer, thereby informing the design of more efficient therapeutics. EV internalization was examined in HeLa cells grown as monolayers and spheroids using flow cytometry and confocal microscopy, with chemical inhibitors and siRNA targeting key proteins of endocytic pathways. Chemical inhibition and siRNA knockdown experiments revealed that EV uptake requires cholesterol, tyrosine kinase, Na⁺/H⁺ exchange, and PI3K activity, and is mediated by caveolin‑1, flotillin‑1, RhoA, Rac1, and PAK1, indicating that EVs predominantly enter cells through clathrin‑independent endocytosis and macropinocytosis.

Abstract

Recent evidence has established that extracellular vesicles (EVs), including exosomes and microvesicles, form an endogenous transport system through which biomolecules, including proteins and RNA, are exchanged between cells. This endows EVs with immense potential for drug delivery and regenerative medicine applications. Understanding the biology underlying EV-based intercellular transfer of cargo is of great importance for the development of EV-based therapeutics. Here, we sought to characterize the cellular mechanisms involved in EV uptake. Internalization of fluorescently-labeled EVs was evaluated in HeLa cells, in 2D (monolayer) cell culture as well as 3D spheroids. Uptake was assessed using flow cytometry and confocal microscopy, using chemical as well as RNA interference-based inhibition of key proteins involved in individual endocytic pathways. Experiments with chemical inhibitors revealed that EV uptake depends on cholesterol and tyrosine kinase activity, which are implicated in clathrin-independent endocytosis, and on Na+/H+ exchange and phosphoinositide 3-kinase activity, which are important for macropinocytosis. Furthermore, EV internalization was inhibited by siRNA-mediated knockdown of caveolin-1, flotillin-1, RhoA, Rac1 and PAK1, but not clathrin heavy chain. Together, these results suggest that EVs enter cells predominantly via clathrin-independent endocytosis and macropinocytosis. Identification of EV components that promote their uptake via pathways that lead to functional cargo transfer might allow development of more efficient therapeutics through EV-inspired engineering.