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.

Extracellular vesicles (EVs) form an endogenous transport system for intercellular transfer of biological cargo, including RNA, that plays a pivotal role in physiological and pathological processes. Unfortunately, whereas biological effects of EV-mediated RNA transfer are abundantly studied, regulatory pathways and mechanisms remain poorly defined due to a lack of suitable readout systems. Here, we describe a highly-sensitive CRISPR-Cas9-based reporter system that allows direct functional study of EV-mediated transfer of small noncoding RNA molecules at single-cell resolution. Using this CRISPR operated stoplight system for functional intercellular RNA exchange (CROSS-FIRE) we uncover various genes involved in EV subtype biogenesis that play a regulatory role in RNA transfer. Moreover we identify multiple genes involved in endocytosis and intracellular membrane trafficking that strongly regulate EV-mediated functional RNA delivery. Altogether, this approach allows the elucidation of regulatory mechanisms in EV-mediated RNA transfer at the level of EV biogenesis, endocytosis, intracellular trafficking, and RNA delivery.

Key Points CLEC-2 activation induces proteolytic cleavage of GPVI and FcγRIIa but not itself. CLEC-2 but not GPVI is detected on platelet-derived microparticles.

Decoration of isolated extracellular vesicles with recombinant phosphatidylserine-binding nanobodies increases their uptake by tumor cells.

The therapeutic use of RNA interference is limited by the inability of siRNA molecules to reach their site of action, the cytosol of target cells. Lipid nanoparticles, including liposomes, are commonly employed as siRNA carrier systems to overcome this hurdle, although their widespread use remains limited due to a lack of delivery efficiency. More recently, nature's own carriers of RNA, extracellular vesicles (EVs), are increasingly being considered as alternative siRNA delivery vehicles due to their intrinsic properties. However, they are difficult to load with exogenous cargo. Here, EV-liposome hybrid nanoparticles (hybrids) are prepared and evaluated as an alternative delivery system combining properties of both liposomes and EVs. It is shown that hybrids are spherical particles encapsulating siRNA, contain EV-surface makers, and functionally deliver siRNA to different cell types. The functional behavior of hybrids, in terms of cellular uptake, toxicity, and gene-silencing efficacy, is altered as compared to liposomes and varies among recipient cell types. Moreover, hybrids produced with cardiac progenitor cell (CPC) derived-EVs retain functional properties attributed to CPC-EVs such as activation of endothelial signaling and migration. To conclude, hybrids combine benefits of both synthetic and biological drug delivery systems and might serve as future therapeutic carriers of siRNA.